title: Drosophila testes, 24h PHS, Polysome fraction '80S', Replicate 2

description: biological replicate 2

value: Drosophila testes

value: bam-/-;hsBam


title: 35S_LZ_45FR_rep1

status: Public on May 14 2024

type: SRA

city: Umeå

first: Stephan

last: Wenkel

address: Parallellvägen 21

country: Sweden

institute: Umeå Plant Science Center

library_source: transcriptomic

data_processing:
BASH command line, FastQC and R
Sequence reads were trimmed for adaptor sequence/low quality sequence using Trimmomatic (Leading = 25, Trailing = 25, MINLEN = 35, Sliding Window = 5:35). Reads quality was checked using FastQC.
Mapping was done using STAR in R
Read counts extraction and normalization were performed in R
The counts from each sample were merged together in a merged count file of that biological replicate, after checking the replicates by PCA
Assembly: TAIR10
Supplementary files format and content: Tab delimited text files combining raw counts of each replicate in one text file per biological sample

molecule: total RNA

organism: Arabidopsis thaliana

source_name: Whole 12 days old seedlings

extract_protocol:
Total RNA was extracted using either the Spectrum Plant Total RNA Kit (Sigma)
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.

tag: tissue

value: Whole 12 days old seedlings

tag: cell line

value: 35::ATHB2:LZ in Col-0 background

tag: cell type

value: Homozygous genomic insertion and overexpression of exons 3 and 4 of ATHB2, controlled by a 35S promoter

tag: genotype

tag: treatment

value: 45 minutes FR-enriched light


title: small RNA-seq: ago18HM-5mm-rep2

city: Wuhan

first: Junpeng

last: Zhan

state: Hubei

address: 1 Shizishan Street, Hongshan District

country: China

institute: Huazhong Agricultural University

data_processing:
To quantify phasiRNA abundance, sRNA-seq reads were mapped to the respective Zea genomes using Bowtie v1.3.1; to quantify miRNA abundance, sRNA-seq reads were mapped to a FASTA file of all mature miRNAs annotated for each genome using bowtie; and to quantify mRNA abundance, Smart-seq2 reads were mapped to the respective Zea genomes (B73 RefGen_v5 was used for the ago18 mutant data) using HISAT v2.1.0. Reads mapped to PHAS loci or miRNAs were counted using featureCounts v1.6.3 and normalized to counts per million (CPM) using edgeR v4.0.2. Differential expression analyses were performed using edgeR with a generalized linear model-based method. Small RNA-transcript interactions in each anther sample were identified using the nanoPARE analysis pipeline (Schon et al., 2018). The sRNA-target pairs with adjusted P value < 0.05 at the EndCut step and are detected in at least two biological replicates were considered positive.
Assembly: Respective reference genomes of Zea varieties
Supplementary files format and content: CPM tables for sRNA-seq and RNA-seq, cleavage-site tables for nanoPARE

organism: Zea mays subsp. mays

source_name: anther

extract_protocol:
Fresh anthers were snap-frozen in liquid nitrogen and total RNA was extracted using the TRI reagent (Sigma-Aldrich) or the TraPR Small RNA Isolation kit (Lexogen; for TraPR sRNA-seq only)
sRNA-seq libraries were prepared from ~100 ng total RNA per sample using a Somagenics RealSeq-AC miRNA library kit following the manufacturer’s protocol. RNA-seq (Smart-seq2) and nanoPARE libraries were prepared from 5 ng total RNA per sample using the nanoPARE library preparation protocol.

tag: group

value: anther

value: mostly B73

tag: molecule subtype

value: small RNA - total


title: 35SLZ_WL_rep2

value: White light


title: Drosophila testes, 24h PHS, Polysome fraction '40S', Replicate 2

city: Stanford

first: Cameron

middle: W

last: Berry

state: California

address: 279 W. Campus Drive, B300

department: Developmental Biology

country: USA

institute: Stanford University

description: biological replicate 2

data_processing:
Sequence reads were mapped to dm6 using BOWTIE version 0.12.8 with a value of e = 5000 to allow for expected mismatches in the poly-A tail not encoded in the genome
Custom perl script written to identify sequencing reads that contain polyA sequences that were not genomically encoded
Reads within annotated 3'UTRs (with 500bp extension) were assigned to the nearest upstream CDS end as defined by Flybase
Assembly: dm6
Supplementary files format and content: tab-delimited text files containing in column: 1) location of CDS end, 2) gene symbol, 3) associated cleavage site location and count (location and count separated by *, and multiple cleavage sites are separate by |)

organism: Drosophila melanogaster

source_name: Drosophila testes

growth_protocol: Progeny of fly crosses were grown at 22C in bottles containing standard molasses media

extract_protocol:
For Polysome profiling, RNA was isolated by acid phenol-chloroform extraction and fractions were combined based on the absorbance profiles at A260 nm to create 6 groups: free, 40S, 60S, 80S, 2-3 ribosomes and 4+ ribosomes
RNA libraries for RNA-seq were prepared using QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen) following manufacturer's protocols.

treatment_protocol: Pupae were heat shocked by placing bottles in 37C water bath for 30 mins and returned to 25C incubator

value: 30 min 37C heat shock

tag: time

value: 24 hours


title: embryo4_cell4

city: Edinburgh

first: Lawrence

middle: E

last: Bates

address: Crewe Road

department: Human Genetics Unit

country: United Kingdom

institute: University of Edinburgh

description: lane1_Ferdinand_Embryos_Plate2_B08_GGATTAGG

library_source: transcriptomic single cell

data_processing:
Raw reads were processed as previously (https://doi.org/10.1038/s41467-018-03149-4), aligning to the GRCh38 reference genome using Hisat2 (https://doi.org/10.1038/s41587-019-0201-4).
Gene expression counts were computed from the mapped data using the featureCounts function within RSubread (https://doi.org/10.1093/nar/gkz114).
Assembly: GRCh38
Supplementary files format and content: Tab-delimited text file with raw counts for each sample

molecule: polyA RNA

organism: Homo sapiens

source_name: Blastocyst

extract_protocol:
Cells were collected directly into 2.5 μl methyltransferase reaction mixture which was comprised of 1 × M.CviPI Reaction buffer, 2 U M.CviPI, 160 μM S-adenosylmethionine, 1 U μl−1 RNAsein, 0.1% IGEPAL then incubated for 15 min at 37 °C. The reaction was stopped and the RNA preserved with the addition of 5 μl RLT plus.
Single-cell RNA sequencing libraries were prepared according to the RNA half of the scNMT-seq protocol (https://doi.org/10.1038/s41467-018-03149-4). Briefly, biotinylated oligo-dT was bound to magnetic beads which were added to single-cell lysates in 96w plates. The captured mRNA was then reverse transcribed and PCR amplified on beads accordingto the Smartseq2 protocol (https://doi.org/10.1038/nprot.2016.138,https://doi.org/10.1038/nprot.2014.006). Indexed Illumina libraries were prepared from the amplified cDNA using Nextera XT reagents and then pooled and sequenced on a HiSeq 2000 instrument using 75bp paired-end reads.

value: Blastocyst

value: ICM

value: WT

tag: developmental stage

tag: Sex

value: Male


title: embryo4_cell3

description: lane1_Ferdinand_Embryos_Plate2_B07_GAGGATGG


title: Drosophila testes, 24h PHS, Polysome fraction 'free', Replicate 2

description: biological replicate 2


title: embryo4_cell2

description: lane1_Ferdinand_Embryos_Plate2_A08_TCAGGAGG


title: Drosophila testes, 72h PHS, total lysate, Replicate 2

description: biological replicate 2

value: 72 hours


title: small RNA-seq: ago18HT-2mm-rep1


title: small RNA-seq: ago18HM-5mm-rep1


title: small RNA-seq: ago18HM-2mm-rep3


title: 35SLZ_WL_rep1


title: 35S_LZ_45FR_rep2


title: embryo4_cell1

description: lane1_Ferdinand_Embryos_Plate2_A07_TACTTCGG


title: Drosophila testes, 48h PHS, total lysate, Replicate 2

description: biological replicate 2

value: Drosophila testes

value: 48 hours


title: small RNA-seq: ago18HM-2mm-rep2


title: embryo3_cell8

description: lane1_Ferdinand_Embryos_Plate2_H05_GTATCGTC

value: Female


title: Drosophila testes, 24h PHS, Polysome fraction '60S', Replicate 2

description: biological replicate 2

value: bam-/-;hsBam


title: t-athb2_90FR_rep1

value: Whole 12 days old seedlings

value: Homozygous insertion causing knockout of ATHB2

value: 90 minutes FR enriched light


title: small RNA-seq: ago18HM-2mm-rep1

value: anther

value: mostly B73


title: small RNA-seq: ago18HM-5mm-rep3

value: small RNA - total


title: t-athb2_45FR_rep3

value: 45 minutes FR-enriched light


title: small RNA-seq: ago18HM-0.5mm-rep3


title: Drosophila testes, 32h PHS, total lysate, Replicate 2

description: biological replicate 2

value: 30 min 37C heat shock

value: 32 hours


title: Drosophila testes, 24h PHS, total lysate, Replicate 2

description: biological replicate 2

value: 24 hours


title: embryo3_cell6

description: lane1_Ferdinand_Embryos_Plate2_F05_GGTAAGAG

value: Blastocyst

value: ICM

value: WT


title: Drosophila testes, 48h PHS, Polysome fraction '4+ ribosomes', Replicate 2

description: biological replicate 2

value: Drosophila testes


title: t-athb2_90FR_rep3


title: small RNA-seq: ago18HM-0.5mm-rep1


title: t-athb2_45FR_rep1


title: Drosophila testes,16h PHS, total lysate, Replicate 2

description: biological replicate 2

value: 16 hours


title: t-athb2_45FR_rep2


title: embryo3_cell5

description: lane1_Ferdinand_Embryos_Plate2_E05_ATTAGCGG

value: Female


title: t-athb2_WL_rep3

value: White light


title: small RNA-seq: ago18HM-0.5mm-rep2


title: Drosophila testes, 72h PHS, Polysome fraction '4+ ribosomes', Replicate 1

description: biological replicate 1

value: bam-/-;hsBam

value: 72 hours


title: t-athb2_WL_rep2

value: Whole 12 days old seedlings

value: Homozygous insertion causing knockout of ATHB2


title: Drosophila testes, 72h PHS, Polysome fraction '2-3 ribosomes', Replicate 1

description: biological replicate 1


title: embryo3_cell4

description: lane1_Ferdinand_Embryos_Plate2_D05_GCTATGAC


title: Drosophila testes, 0h PHS, total lysate, Replicate 2

description: biological replicate 2

value: 0 hours


title: t-athb2_90FR_rep2

value: 90 minutes FR enriched light


title: small RNA-seq:Il14H-2mm-rep4

value: anther

value: small RNA - total


title: embryo3_cell3

description: lane1_Ferdinand_Embryos_Plate2_C06_CTTGCTTC


title: small RNA-seq:Il14H-2mm-rep3


title: Drosophila testes, 72h PHS, Polysome fraction '80S', Replicate 1

description: biological replicate 1

value: 30 min 37C heat shock


title: small RNA-seq:Il14H-0.4mm-rep5


title: embryo3_cell2

description: lane1_Ferdinand_Embryos_Plate2_B06_GCTCCTTG


title: embryo3_cell1

description: lane1_Ferdinand_Embryos_Plate2_A06_TTGGTATG


title: WT_90FR_rep3

value: Wild-type seedlings


title: small RNA-seq:Il14H-0.4mm-rep4


title: embryo3_cell7

description: lane1_Ferdinand_Embryos_Plate2_G05_GGTGTCGA

value: Blastocyst


title: t-athb2_WL_rep1


title: Drosophila testes, 72h PHS, Polysome fraction '60S', Replicate 1

description: biological replicate 1


title: embryo2_cell10

value: ICM

value: Male


title: Drosophila testes, 72h PHS, Polysome fraction '40S', Replicate 1

description: biological replicate 1

value: Drosophila testes


title: WT_90FR_rep2

value: WT


title: small RNA-seq:Il14H-0.4mm-rep3


title: embryo2_cell9


title: small RNA-seq: Oh43-2mm-rep2


title: embryo2_cell8


title: Drosophila testes, 48h PHS, Polysome fraction '4+ ribosomes', Replicate 1

description: biological replicate 1

value: 48 hours


title: WT_90FR_rep1


title: embryo2_cell7


title: WT_45FR_rep3

value: 45 minutes FR-enriched light


title: WT_45FR_rep2


title: small RNA-seq: Oh43-2mm-rep3


title: embryo2_cell6


title: WT_45FR_rep1


title: small RNA-seq: Oh43-0.4mm-rep2


title: small RNA-seq: Oh43-2mm-rep1


title: embryo2_cell5


title: Drosophila testes, 48h PHS, Polysome fraction '80S', Replicate 1

description: biological replicate 1

value: bam-/-;hsBam


title: Drosophila testes, 48h PHS, Polysome fraction '60S', Replicate 1

description: biological replicate 1


title: embryo2_cell4


title: small RNA-seq: Oh43-0.4mm-rep1


title: small RNA-seq: NC358-2mm-rep3


title: WT_WL_rep2

value: White light


title: Drosophila testes, 48h PHS, Polysome fraction '40S', Replicate 1

description: biological replicate 1


title: embryo2_cell3


title: embryo2_cell2


title: Drosophila testes, 72h PHS, Polysome fraction 'free', Replicate 1

description: biological replicate 1

value: 72 hours


title: Drosophila testes, 24h PHS, Polysome fraction '4+ ribosomes', Replicate 1

description: biological replicate 1

value: 24 hours


title: small RNA-seq: NC358-2mm-rep2


title: pancreatic rat islets, anti-mt-tRF-LeuTAA_6, adult 3-month-old, Wistar

city: Lausanne

first: Cecile

last: Jacovetti

address: Rue du Bugnon 9

department: Fundamental Neurosciences

country: Switzerland

institute: University of Lausanne

data_processing:
Purity-filtered reads were adapters and quality trimmed with Cutadapt (v. 1.8, Martin 2011). Reads matching to ribosomal RNA sequences were removed with fastq_screen (v. 0.11.1). . To determine transcript expression levels, reads were aligned against the Rnor_6.0.98 genome using STAR (v. 2.5.3a).
The number of read counts per gene locus was summarized with HTseq-count (v. 0.9.1) using Rnor_6.0.98 gene annotation.
Quality of the RNA-sequencing data alignment was assessed using RSeQC (v. 2.3.7).
Normalization and differential gene expression (FDR ≤ 0.05) were performed in R (R version 4.0.3).
Genes with low counts were filtered out using the function filterByExpr with default parameters (limma v 3.44.3): this keeps genes that have ³ 10 counts in at least n = 6 samples and ³ 15 counts summed across all samples. Library sizes were then scaled using trimmed mean of M values  (TMM) normalization. Subsequently, the normalized counts were transformed to counts per million (cpm) values and a log2 transformation was applied by means of the function cpm with the parameter setting prior counts = 1. (EdgeR v 3.30.3; Robinson et al. 2010). Differential expression was computed with the R Bioconductor package Limma (Ritchie et al. 2015) by fitting data to a linear model. The approach Limma-trend was used.
Assembly: Alignment to the genome using HTSeq: Reads were aligned against the Rnor_6.0.98 genome using STAR. Quality of the RNA-seq data alignment was assessed using RSeQC.
Supplementary files format and content: detected genes upon mt-tRF-LeuTAA silencing vs anti-control.xlsx
Supplementary files format and content: up and down genes upon mt-tRF-LeuTAA silencing vs anti-control.xlsx

organism: Rattus norvegicus

source_name: dispersed pancreatic islets

growth_protocol: Rat pancreatic islets were isolated by collagenase digestion followed by Histopaque density gradient and handpicking. Islets were dispersed into single cells by incubation in Ca2+/Mg2+ free phosphate-buffered saline, 3 mM EGTA, and 0.002 % trypsin (Thermo Fischer Scientific, # 15400-054) for 3-4 min at +37 °C. Isolated rodent islets as well as dispersed islet cells were cultured in RPMI 1640 GlutaMAX medium (Thermo Fischer Scientific # 72400-054) containing 11 mM glucose and 2 mM L-glutamine and supplemented with 10 % fetal calf serum (Merck-Sigma # F7524), 10 mM Hepes pH 7.4, 1 mM sodium pyruvate (Merck-Sigma # S-8636), 100 mg/mL streptomycin and 100 IU/mL penicillin.

extract_protocol:
Total RNA from rat islets was extracted using miRNeasy micro kit (Qiagen # 217084).
TruSeq Stranded Total RNA kit was used to prepare librairies.

treatment_protocol: Rat islet cells were transfected with 30 picomoles single stranded antisense oligonucleotides that correspond to the complementary sequence of the targeted fragment mt-tRF-LeuTAA (Qiagen # custom power inhibitor PS desalted, anti-mt-tRF-LeuTAA) or scrambled control oligonucleotides (anti-control) using Lipofectamine 2000 (Thermo Fischer Scientific # 11668019). Cells were then cultured for 72h before RNA extraction.

value: pancreatic islets

value: adult

tag: strain

value: Wistar

value: antisense against mt-tRF-LeuTAA (anti-mt-tRF-LeuTAA) oligonucleotides


title: small RNA-seq: NC358-2mm-rep1

value: anther

value: small RNA - total


title: Drosophila testes, 48h PHS, Polysome fraction 'free', Replicate 1

description: biological replicate 1

value: 30 min 37C heat shock


title: small RNA-seq: NC358-0.4mm-rep2


title: pancreatic rat islets, anti-mt-tRF-LeuTAA_5, adult 3-month-old, Wistar


title: Drosophila testes, 24h PHS, Polysome fraction '80S', Replicate 1

description: biological replicate 1


title: embryo2_cell1

value: Blastocyst

value: ICM


title: embryo1_cell10

value: Female


title: embryo1_cell9

value: WT


title: pancreatic rat islets, anti-mt-tRF-LeuTAA_3, adult 3-month-old, Wistar


title: WT_WL_rep3

value: Whole 12 days old seedlings

value: Wild-type seedlings


title: Drosophila testes, 24h PHS, Polysome fraction '60S', Replicate 1

description: biological replicate 1

value: Drosophila testes

value: bam-/-;hsBam


title: small RNA-seq: NC358-0.4mm-rep1


title: embryo1_cell8


title: small RNA-seq: HP301-2mm-rep2


title: pancreatic rat islets, anti-mt-tRF-LeuTAA_2, adult 3-month-old, Wistar


title: small RNA-seq: HP301-2mm-rep1


title: Drosophila testes, 24h PHS, Polysome fraction '40S', Replicate 1

description: biological replicate 1


title: pancreatic rat islets, anti-mt-tRF-LeuTAA_1, adult 3-month-old, Wistar


title: Drosophila testes, 24h PHS, Polysome fraction 'free', Replicate 1

description: biological replicate 1


title: WT_WL_rep1

value: White light


title: embryo1_cell7


title: small RNA-seq: HP301-0.4mm-rep2


title: embryo1_cell6


title: Drosophila testes, 24h PHS, Polysome fraction '2-3 ribosomes', Replicate 1

description: biological replicate 1

value: 24 hours


title: pancreatic rat islets, anti-control_6, adult 3-month-old, Wistar

value: pancreatic islets

value: antisense control (anti-control) oligonucleotides


title: small RNA-seq: HP301-0.4mm-rep1


title: Drosophila testes, 72h PHS, total lysate, Replicate 1

description: biological replicate 1

value: 72 hours


title: embryo1_cell5


title: pancreatic rat islets, anti-mt-tRF-LeuTAA_4, adult 3-month-old, Wistar

value: adult

value: Wistar

value: antisense against mt-tRF-LeuTAA (anti-mt-tRF-LeuTAA) oligonucleotides


title: Drosophila testes, 32h PHS, total lysate, Replicate 1

description: biological replicate 1

value: 30 min 37C heat shock

value: 32 hours


title: embryo1_cell4

value: Blastocyst

value: ICM


title: embryo1_cell3

value: WT

value: Female


title: small RNA-seq: B73-0.4mm-rep2

value: anther

value: small RNA - total


title: pancreatic rat islets, anti-control_5, adult 3-month-old, Wistar


title: Drosophila testes, 48h PHS, total lysate, Replicate 1

description: biological replicate 1

value: Drosophila testes

value: bam-/-;hsBam

value: 48 hours


title: small RNA-seq: B73-2mm-rep2


title: pancreatic rat islets, anti-control_4, adult 3-month-old, Wistar

value: pancreatic islets


title: Drosophila testes,16h PHS, total lysate, Replicate 1

description: biological replicate 1

value: 16 hours


title: Drosophila testes, 24h PHS, total lysate, Replicate 1

description: biological replicate 1

value: 24 hours


title: pancreatic rat islets, anti-control_2, adult 3-month-old, Wistar

value: adult

value: antisense control (anti-control) oligonucleotides


title: pancreatic rat islets, anti-control_3, adult 3-month-old, Wistar

value: Wistar


title: embryo1_cell2


title: pancreatic rat islets, anti-control_1, adult 3-month-old, Wistar


title: embryo1_cell1


title: Drosophila testes, 0h PHS, total lysate, Replicate 1

description: biological replicate 1

value: 30 min 37C heat shock

value: 0 hours


title: small RNA-seq: B73-0.4mm-rep1


title: genomic DNA from M0 macrophage with spermine

status: Public on May 13 2024

type: genomic

city: Hangzhou

first: Hongde

last: Li

state: Zhejiang

address: 150 Fucheng Road

institute: Hangzhou Institute of Medicine, Chinese Academy of Sciences

description: Spermine

hyb_protocol: Bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium Human MethylationEPIC Beadchip using standard Illumina protocol

scan_protocol: Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting

data_processing: GenomeStudio

label: Cy3 and Cy5

molecule: genomic DNA

source_name: Primary macrophage

label_protocol: Standard Illumina Protocol

growth_protocol: Cells were cultured in RPMI1640 medium supplemented with 10% FBS.

extract_protocol: Genomic DNA was extracted and purified from cell samples using Qiagen DNeasy Kit according to the instructions

treatment_protocol: Treated with 10 μM spermine or 30% MDA-MB-231 conditioned media fro 24 hours

value: Primary macrophage


title: genomic DNA from TAMs induced by MDA-MB-231 cell-conditioned medium

description: TAM


title: genomic DNA from M0 macrophage with PBS

description: Control


title: genomic DNA from normal M0 macrophage


title: Drosophila testes, 48h PHS, Polysome fraction '2-3 ribosomes', Replicate 1

description: biological replicate 1


title: FtL knockout LPS 3 [FL.LM71]

city: Birmingham

first: SUBHASHINI

last: BOLISETTY

state: AL

address: 1918 University Blvd,, MCLM 523

institute: University of Alabama at Birmingham

description: Total RNA extraction followed by poly A selection and library preparation (Azenta).

data_processing:
Sequence reads were trimmed for adaptor sequence/low-quality sequenc using Trim_Galore
Trimmed sequence reads were mapped to mm10 using Star
Read count extraction was performed with featurecounts of the subread package
Raw counts were normalized using Deseq2
Assembly: mm10
Supplementary files format and content: normalized counts are the Deseq2 median of ratios

organism: Mus musculus

source_name: Kidney

extract_protocol:
Total RNA was extracted from a 1/2 kidney using trireagent and the Zymo Direct-zol RNA mini kit
RNA underwent polyA selection and then cDNA library synthesis with Illumina kits.

treatment_protocol: 10-14 week old mice were either left untreated, or treated with LPS (8mg/kg bw) and kidneys collected after 24 hours.  1/2 kidney was cut into small 5 mm cubes and snap frozen

value: Kidney

value: FtL knockout

value: LPS treatment for 24 hours


title: FtL knockout LPS 5 [FL.LM91]


title: FtL knockout LPS 2 [FL.LM111]


title: FtL knockout LPS 4 [FL.LM81]


title: small RNA-seq: B73-2mm-rep1

value: anther


title: C. albicans_60min_Control_R2

city: Montreal

first: Adnane

last: Sellam

address: 5000, rue Bélanger

country: Canada

institute: Universite de Montreal

data_processing:
For RNA-seq, sequencing reads were filtered with fastp version 0.23.2.
For RNA-seq, filtered reads were mapped to the reference genome sequence of strain SC5314 and C. auris 381 with STAR version 2.7.9a
For RNA-seq, the number of reads mapping on each of the genes was calculated by featurecount version 2.0.1.  For the drug-induced haploinsufficiency profiling , unique uptag DNA barcodes were extracted and counted using SeqKit
Differential gene expression and barcode abundance were calculated with DESeq2 version 1.26.0.
Assembly: Candida albicans assembly 21 and Candida auris B8441
Supplementary files format and content: "raw_counts_SPB00525_C.albicans.txt" file contains read counts for each experimental condition and replicate
Supplementary files format and content: "raw_counts_SPB00525_C.auris.txt" file contains read counts for each experimental condition and replicate
Supplementary files format and content: "Mutant fitness scores.txt" file contains barcode counts of each mutant in each experimental condition and replicate

organism: Candida albicans SC5314

source_name: WT control C. albicans cells in SC medium for 60min_rep2

growth_protocol: For RNA-seq profiling, overnight cultures of C. albicans SC5314 and C. auris 381 strains were diluted to an OD600 of 0.1 in 50 ml of fresh SC medium and grown at 30°C under agitation (200 rpm) to early logarithmic phase (OD600=0.6).

extract_protocol:
For RNA-seq, total RNA was extracted using an RNAeasy purification kit (Qiagen) and glass bead lysis in a Biospec Mini 24 bead-beater. For the drug-induced haploinsufficiency profiling, cells were harvested by centrifugation and genomic DNA was extracted using YeaStar kit (Zymo Research).
For RNA-seq, the NEBNext UltraTM II RNA Library Prep Kit for Illumina was used to construct the RNA-seq library. For the drug-induced haploinsufficiency profiling, the Up-tag DNA-barcodes were amplified using 25 ng genomic DNA with DBC-F1 and DBC-R1 primers that recognize the common region of up-tag barcode and contain the multiplexing tag (DBC-F1) and sequences required for hybridization to the Illumina ﬂow cell. PCR amplification products were purified form an agarose gel using the QIAquick Gel Extraction kit (Qiagen) and quantiﬁed by QuantiFluor dsDNA System (Promega). Equal quantity of DNA from the SPB00525-treated and non-treated pools were combined prior to NGS sequencing using Illumina MiSeq platform and DBC1 and DBC2 sequencing.

treatment_protocol: For RNA-seq, cultures were then either left untreated or exposed to  6 µg/ml SPB00525 and incubated at 30°C for 15- and 60-min. Cells were harvested by centrifugation and were flash-frozen and stored at -80°C. For each condition, a total of two biological replicates were considered for RNA-seq analysis. For the drug-induced haploinsufficiency profiling, the double-barcoded heterozygote C. albicans (DBC) mutant pools were diluted to OD600 = 0.062 and exposed or not to 6 µg/ml SPB00525 in either SC or YPD and grown at 30°C under agitation for 15 hours. 1 mL of the sub-cultured pool was distributed into triplicate culture tubes, each containing 1 mL of YPD or SC medium supplemented with either 6 µg/ml SPB00525 or DMSO solvent. These cultures were incubated at 30°C under shaking conditions for 24 hours.

value: Yeast cells

value: WT

value: 60 min in SC medium


title: C. albicans_60min_SPB00525 _R2

source_name: WT control C. albicans cells in SC medium treated with SPB00525 for 60 min_rep2

value: 60 min in SC medium + 6 ug/ml SPB00525


title: C. albicans_60min_SPB00525 _R1

source_name: WT control C. albicans cells in SC medium treated with SPB00525 for 60 min_rep1


title: C. albicans_15min_SPB00525 _R2

source_name: WT control C. albicans cells in SC medium treated with SPB00525 for 15 min_rep2

value: 15 min in SC medium + 6 ug/ml SPB00525


title: C. albicans_60min_Control_R1

source_name: WT control C. albicans cells in SC medium for 60min_rep1


title: FtL knockout LPS 1 [FL.LM101]


title: C. albicans_15min_Control_R2

source_name: WT control C. albicans cells in SC medium for 15min_rep2

value: 15 min in SC medium


title: Wildtype LPS 4 [FtL.LM4]

value: Wildtype


title: C. albicans_15min_Control_R1

source_name: WT control C. albicans cells in SC medium for 15min_rep1


title: SPB00525_SC_rep1_R2

library_source: genomic

data_processing:
For RNA-seq, sequencing reads were filtered with fastp version 0.23.2.
For RNA-seq, filtered reads were mapped to the reference genome sequence of strain SC5314 and C. auris 381 with STAR version 2.7.9a
For RNA-seq, the number of reads mapping on each of the genes was calculated by featurecount version 2.0.1.  For the drug-induced haploinsufficiency profiling , unique uptag DNA barcodes were extracted and counted using SeqKit
Differential gene expression and barcode abundance were calculated with DESeq2 version 1.26.0.
Assembly: Candida albicans assembly 21 and Candida auris B8441
Supplementary files format and content: "raw_counts_SPB00525_C.albicans.txt" file contains read counts for each experimental condition and replicate
Supplementary files format and content: "raw_counts_SPB00525_C.auris.txt" file contains read counts for each experimental condition and replicate
Supplementary files format and content: "Mutant fitness scores.txt" file contains barcode counts of each mutant in each experimental condition and replicate
Library strategy: Barcode-seq

source_name: Pooled C. albicans heterozygous mutants in SC medium treated with SPB00525_rep2

value: Heterozygous mutants

value: SC medium +  6 ug/ml SPB00525


title: Wildtype LPS 3 [FtL.LM3]


title: Wildtype LPS 5 [FtL.LM7]


title: Wildtype LPS 2 [FtL.LM2]


title: SPB00525_SC_rep1_R1

source_name: Pooled C. albicans heterozygous mutants in SC medium treated with SPB00525_rep1


title: FtL knockout baseline 5 [FL.BM81]

value: untreated


title: SC_rep1_R2

source_name: Pooled C. albicans heterozygous mutants in SC medium_rep2

value: SC medium


title: Wildtype LPS 1 [FtL.LM1]


title: SC_rep1_R1

source_name: Pooled C. albicans heterozygous mutants in SC medium_rep1


title: SPB00525_YPD_rep1_R2

source_name: Pooled C. albicans heterozygous mutants in YPD medium treated with SPB00525_rep2

value: YPD medium +  6 ug/ml SPB00525


title: FtL knockout baseline 4 [FL.BM71]


title: FtL knockout baseline 3 [FL.BM61]


title: YPD_rep1_R2

source_name: Pooled C. albicans heterozygous mutants in YPD medium_rep2

value: YPD medium


title: FtL knockout baseline 2 [FL.BM41]

value: Kidney


title: YPD_rep1_R1

source_name: Pooled C. albicans heterozygous mutants in YPD medium_rep1


title: C. albicans_15min_SPB00525 _R1

source_name: WT control C. albicans cells in SC medium treated with SPB00525 for 15 min_rep1


title: Wildtype baseline 2 [FtL.BM3]


title: Wildtype baseline 4 [FtL.BM5]


title: Wildtype baseline 5 [FtL.BM6]


title: Wildtype baseline 3 [FtL.BM4]


title: FtL knockout baseline 1 [FL.BM11]

value: FtL knockout


title: treated_with_loaded_MN_rep3

city: Boston

first: Jamil

middle: R

last: Azzi

state: MA

address: 221 Longwood ave

department: Transplantation Research Center

institute: Brigham and Women's Hospital

data_processing:
raw reads were aligned to mouse reference genome using Hisat2
raw counts matrix was generated using FeatureCounts
Assembly: mm39
Supplementary files format and content: tabulated format containing the raw counts for each sample

source_name: Skin

extract_protocol:
RNA was extracted with Qiagen miRNeasy kit.
Libraries were prepared for RNA-Seq using IDT’s xGen Broad-Range RNA Library Prep Kit, according to manufacturer’s directions.

value: Skin

value: treated with IL-2+CCL22 loaded MNs


title: untreated_rep2


title: untreated_rep1


title: treated_with_empty_MN_rep3

value: treated with empty MN


title: treated_with_empty_MN_rep2


title: treated_with_loaded_MN_rep2


title: treated_with_loaded_MN_rep1


title: PFC_Ttr-KD, rep4

city: Jena

first: Konstantin

last: Riege

address: Beutenbergstraße 11

country: Germany

institute: Leibniz Institute on Aging - Fritz Lipmann Institute (FLI)

data_processing:
Sequence information was converted to FASTQ format using bcl2fastq v2.20.0.422
Raw data was quality trimmed and clipped for Illumina universal adapter. Potential sequencing errors were detected and corrected using Rcorrector and rRNA fragments have been depleted using SortMeRNA.
Reads were mapped on GRCm38 (mm10) genome reference using segemehl, deduplicated for over-amplified PCR fragments via Unique Molecular Identifiers using UMI-tools and quantified on Ensembl v102 derived gene annotation utilizing featureCounts. Variance stabilized counts were inferred by DESeq2.
Assembly: mm10 (Ensembl v102)
Supplementary files format and content: Tab delimited text files include raw, variance stabilized and TPM normalized read counts for each sample.

source_name: Brain

growth_protocol: The mice were bred and provided with standard chow (LASQCdiet Rod16-R, LASvendi GMBH, Soest, Germany), containing 50 mg/kg of vitamin B12. Housing conditions involved a 14L:10D light cycle.

extract_protocol:
RNA extraction involved resuspension in Trizol and chloroform precipitation, followed by washing with isopropanol and 75% ethanol. Subsequently, cDNA conversion was performed using the GoScript™ Reverse Transcriptase kit (#A5001, Promega).
Libraries were prepared from 500 ng of input material (total RNA) using NEBNext Ultra II Directional RNA Library Preparation Kit in combination with NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA) following the manufacturer's instructions (NewEngland Biolabs).

treatment_protocol: Bilateral stereotaxic surgery into the dorsomedial PFC was essentially performed as described (doi: 10.1038/nm.4386). Viruses were injected at a rate of .1 μL/min for 5 min using 10 μL Gastight Hamilton syringes model 1801 RN. Mice were scored for general health and behavior at least 3 days post-surgery. The following three viruses were utilized: pAAV.1-CAG-GFP (#37825, Addgene), OE-Ttr-GFP: pAAV-CAG-GFP-P2A-Ttr-WPRE.bgH (custom-made by Charitè viral vector core, Berlin, Germany), KD-Ttr-GFP: pAAV-U6-Ttr-shRNA-2-CAG-GFP-WPRE3 (custom-made by Charitè viral vector core, Berlin, Germany).

value: Brain

tag: region

value: Prefrontal cortex

value: Ttr-KD

value: male


title: PFC_Ttr-KD, rep3


title: treated_with_empty_MN_rep1


title: SPB00525_YPD_rep1_R1

source_name: Pooled C. albicans heterozygous mutants in YPD medium treated with SPB00525_rep1

value: Yeast cells


title: untreated_rep3


title: PFC_Ttr-KD, rep2


title: PFC_Ttr-KD, rep1


title: PFC_Ttr-OE, rep4

value: Ttr-OE


title: Wildtype baseline 1 [FtL.BM2]

source_name: Kidney

value: Wildtype


title: PFC_Ttr-OE, rep3


title: PFC_Ttr-OE, rep2


title: 2M-rep2, C2C12, S4

city: Milan

first: Chiara

last: Lanzuolo

address: Via Francesco Sforza 35

country: Italy

institute: CNR and Istituto Nazionale Genetica Molecolare

data_processing:
Trimming. High-throughput sequencing reads were trimmed using Trimmomatic (v0.39) (Bolger, Lohse, and Usadel 2014) using the following parameters: 2 for seed_mismatch, 30 for palindrome_thresh old, 10 for simple_threshold, 3 for leading, 3 for trailing and 4:15 for sliding window. The sequence minimum length threshold of 35 was applied to all data sets . We used the Trimmomatic “TruSeq3 - SE.fa” (for single end).
Alignment. After trimming, reads were aligned using BWA (v0.7.17 - r1188) (Li and Durbin 2009) setting – k parameter as 2 and using as reference genome the UCSC mm9 genomeand the output saved in BAM file format.
Filtering. The PCR duplicates were marked with Picard (v2.22; https://github.com/broadinstitute/picard) MarkDupuplicates option , then filtered using Samtools (v1.9) (Li et al. 2009). In addition, we filtered all the reads with mapping quality lower than 1. Each sequencing lane was analyzed separately and then merged at the end of the process.
Genomic tracks. To compute reads distribution profile (genomic tracks) we used Deeptools (v3.4.3) (Ramirez et al. 2016) bam Coverage function. For these analyses the genome was binned at 50bp, the reads extended up to 250 bp and RPKM normalization method was used. We considered a genome size of 2308125349 bp  and we excluded regions known to be problematics in term of sequencing reads coverage using the black list f rom the following work : https://doi.org/10.1038/s41598-019-45839-z
Assembly: mm9
Supplementary files format and content: Bigwiggle files (.bw) are created using Deeptools for genomic track data
Library strategy: SAMMY-seq

growth_protocol: Cells were cultured at 37 °C, 5% CO2 in DMEM high glucose supplemented with 10% FBS

extract_protocol:
For 4f-SAMMY-seq, 2 million C2C12 myoblasts were detached from the culture plate by 3min incubation in trypsin-EDTA at 37°C, 5% CO2. After two washes in cold PBS, they were resuspended in 600μL of CSK-Triton buffer (10mM PIPES pH 6.8, 100mM NaCl, 1mM EGTA, 300mM Sucrose, 3mM MgCl2, 1mM PMSF, 1 mM DTT, 0.5% Triton X-100, with protease inhibitors). After 10min incubation on a wheel at 4°C, soluble proteins and the cytoskeletal structure were separated from the nuclei by centrifugation at 900g for 3min at 4°C; the supernatant was labeled as S1 fraction. The pellet was then washed with an additional volume of CSK-Triton buffer, resuspended in 100μL of CSK buffer (10mM PIPES pH 6.8, 100mM NaCl, 1mM EGTA, 300mM Sucrose, 3mM MgCl2, 1mM PMSF, with protease inhibitors) and incubated for 60min at 37°C with 25U of RNase–free DNase I (Invitrogen, AM2222). To stop DNA digestion, ammonium sulphate was added in the CSK buffer to a final concentration of 250mM. After 5min incubation on ice, the sample was pelleted at 900g for 3min at 4°C; the supernatant, containing digested chromatin fragments, was labeled as S2 fraction. Afterwards, the pellet was washed with 200μL of CSK buffer, resuspended in 100μL of CSK-NaCl buffer (CSK buffer with NaCl final concentration increased to 2M) and then incubated 10min on a wheel at 4°C. At the end of the incubation, the sample was centrifuged at 2300g for 3min at 4°C and the supernatant was labeled as S3 fraction. Finally, after two washes in 200μL of CSK-NaCl buffer, the pellet was solubilized in 100μL of 8M urea; the final suspension was labeled as S4 fraction. Half volume of S2 and the the whole volume of S3 and S4 were diluted in TE (10mM TrisHCl pH 7.5, 1mM EDTA pH 8.0) to a final volume of 200μl and then incubated 90min at 37°C with 6μL of RNAse cocktail (Ambion, AM2286), followed by 150min at 55° with Proteinase K (Invitrogen, AM 2548) to a final concentration of 0.2 μg/μL. Next, DNA was isolated through phenol:chloroform:isoamyl alcohol (Sigma, 77617) extraction, precipitated in 70% ethanol, 0.3 M sodium acetate and 20 μg glycogen overnight at -20°C or 1 hour in dry ice and resuspended in nuclease-free water. S2 was additionally purified using PCR DNA Purification Kit (Qiagen, 28106) and then DNA fragments in this fraction were separated using AMPure XP paramagnetic beads (Beckman Coulter, A63880) to obtain S2S (<300bp) and S2L (> 300bp) fractions. In detail, beads were added to the S2 fraction in a 0.95x (v/v) ratio, so to bind fragments larger than 300bp while leaving smaller fragments in solution. Magnetic separation of beads from supernatant allowed the physical separation of larger fragments (on the beads) from shorter ones (in the supernatant). Larger fragments bound on the beads were then washed in 85% ethanol, resuspended in water and magnetically separated from the beads (S2L fraction). Shorter fragments in the supernatant of the first step were bound to beads by adding a further 0.85x (v/v) beads ratio to the suspension; after washing in 85% ethanol and resuspending in water, they were also detached from the beads (S2S fraction). After DNA isolation, S2L, S3 and S4 fractions were transferred to screw cap microTUBEs (Covaris, 004078) and sonicated in a Covaris M220 focused-ultrasonicator to obtain a smear of DNA fragments peaking at 200bp (settings: water bath 20°C, peak power 30.0, duty factor 20.0, cycles/burst 50; duration: 125sec for S2 and S2L, 175sec for S3 and S4). DNA in the fractions was then quantified using Qubit dsDNA HS Assay Kit (Invitrogen, Q32854) and a Qubit 4.0 fluorometer; quality control was performed by run on an Agilent 2100 Bioanalyzer System using the High Sensitivity DNA Kit (Agilent, 5067-4626).
Libraries were created from each fraction using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) and the Unique Dual Index NEBNext Multiplex Oligos for Illumina (NEB, E6440S).

treatment_protocol: No treatment has been performed on these cells.

value: Myoblasts

tag: number of_cells

tag: fraction


title: 2M-rep2, C2C12, S2S


title: 4T1 tumour cells, replicate 2

type: RNA

city: Barcelona

first: Alexandra

last: Avgustinova

address: C/ Baldiri Reixac 10

country: Spain

institute: IRB Barcelona

description:
replicate 2
4T1_T2

hyb_protocol: Standard Illumina hybridisation protocol

scan_protocol: Standard Illumina scanning protocol

data_processing: The data were normalised using quantile normalisation with IlluminaGUI in R. Probes that were not expressed in any of the samples were removed before normalisation to avoid adding noise to the data.

label: Biotin

source_name: FACS isolated, ex vivo

label_protocol: Biotinylated cRNA were prepared using Ambion TotalPrep 96-RNA amplification kit for Illumina arrays

growth_protocol: 5 x 10^5 tumour cells were inoculated orthotopically into the 4th mammary fat pad of Ub-GFP Balb/c mice. Tumours were left to grow for 2-4 weeks, until they reached an average diameter of 10 mm.

extract_protocol: RNA was extracted with Trizol reagen in accordance with the manufacturer´s. Quality control was performed with Agilent Bioanalyser.

treatment_protocol: cells were not treated

value: Balb/c

value: primary tumour

tag: gender

value: female


title: 4T1 tumour cells, replicate 1

description:
replicate 1
4T1_T1


title: 4T07 tumour cells, replicate 3

description:
replicate 3
4T07_T3


title: PFC_ctrl, rep3

value: ctrl


title: 4T1 tumour cells, replicate 3

description:
replicate 3
4T1_T3


title: PFC_ctrl, rep4


title: 67NR tumour cells, replicate 3

description:
replicate 3
67NR_T3


title: 67NR tumour cells, replicate 2

description:
replicate 2
67NR_T2


title: 2M-rep1, C2C12, S3


title: 4T07 tumour cells, replicate 2

description:
replicate 2
4T07_T2


title: PFC_ctrl, rep1


title: 4T07 tumour cells, replicate 1

description:
replicate 1
4T07_T1


title: 2M-rep2, C2C12, S2L


title: 2M-rep1, C2C12, S4


title: 2M-rep1, C2C12, S2S


title: 67NR tumour cells, replicate 1

description:
replicate 1
67NR_T1


title: 2M-rep1, C2C12, S2L


title: One-year-old Islets - Islet 845 knockout

city: Duarte

first: Hsun

middle: Teresa

last: Ku

state: CA

address: 1500 E. Duarte Road

department: Translational Research and Cellular Therapeutics

institute: City of Hope

data_processing:
Illumina NovaSeq6000
RNA-Seq reads were trimmed to remove sequencing adapters using Trimmomatic and polyA tails using FASTP.
The processed reads were mapped to the mouse genome mm10 using STAR (v. 2.6.0.a), and gene expression levels were summarized by HTSeq-count v.0.11.1.
Gene expression raw counts were normalized using TMM normalization method and differential expression analysis was conducted using log likelihood ratio test implemented in “edgeR”.
Assembly: mm10
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample

source_name: Islets

growth_protocol: Control (Tff2fl/fl) and conditional Tff2 knockout (Pdx1:Cre;Tff2fl/fl) male mice were aged until 60 weeks of age (one-year-old). The pancreas was perfused with Collagenase V solution followed by islet isolation using density gradient centrifugation method. Isolated islets were washed and manually picked to reduce contamination from exocrine tissue. Manually picked islets from each mouse were pelleted in 1.5 mL tubes, lysed with 1 mL of RLT buffer containing β-mercaptoethanol and stored at -80 degree freezer prior to RNA extraction and DNase treatment for bulk mRNA-sequencing.

extract_protocol:
RNA was extracted using the RNeasy Micro Kit (Qiagen, Valencia, CA) to obtain 0.5-1 ug total RNA from each sample.
RNA sequencing libraries were prepared with Kapa mRNA HyperPrep kit (Kapa Biosystems) according to the manufacturer's protocol.

value: 60 weeks

value: Islets


title: One-year-old Islets - Islet 812 knockout


title: One-year-old Islets - Islet 122 knockout


title: PFC_Ttr-OE, rep1


title: One-year-old Islets - Islet 858 control


title: HK_24h_pIC_High-w6_T6_2

city: St. John's

first: Albert

last: Caballero Solares

state: NL

address: 0 Marine Lab Road

department: Ocean Sciences

institute: Memorial University of Newfoundland

description: Challenged, 24 h post-injection, High-w6-fed fish

data_processing: The relative quantity (RQ) of each transcript was calculated using the ViiA 7 Software Relative Quantification Study Application (Version 1.2.3; Applied Biosystems). For each GOI, the RQ calculation considered the transcript levels of both normalizer genes for the normalisation, as well as the amplification efficiency, and set the lowest normalized expression value as the calibrator (i.e., assigned an RQ of 1.0).

label: SYBR Green

organism: Salmo salar

source_name: Head kidney

label_protocol:
First-strand cDNA templates for RT-qPCR were synthesized in 20 μL reactions from 1 μg of DNaseI-treated, column-purified total RNA using random primers (250 ng; Invitrogen), dNTPs (0.5 mM final concentration; Invitrogen), and M-MLV reverse transcriptase (200 U; Invitrogen) with the manufacturer’s first strand buffer (1X final concentration) and DTT (10 mM final concentration) at 37°C for 50 min.
PCR amplifications were performed in 13 μL reaction volumes containing 1X Power SYBR Green PCR Master Mix (Applied Biosystems/Life Technologies), 50 nM of both the forward and reverse primers, and the indicated cDNA quantity (see below). The PCR program consisted of 1 cycle of 50°C for 2 min, 1 cycle of 95°C for 10 min and 40 cycles of 95°C for 15 sec and 60°C for 1 min, with fluorescence detection at the end of each 60°C step and was followed by dissociation curve analysis.
Primer amplification performance was tested using the 7500 Fast Real Time PCR system (Applied Biosystems). Single product amplification and absence of primer-dimer in the no-template control (NTC) were assessed via dissociation curve analysis. Amplicon size was checked electrophoretically on 2% agarose gels and compared with a 1 kb Plus DNA Ladder (Invitrogen). Amplification efficiencies were determined based on a 5-point 1:3 dilution series starting with cDNA representing 10 ng of input total RNA.
RT-qPCR analyses of expression levels of 46 genes of interest (GOI) and 2 normalizer genes were performed using the ViiA 7 Real Time PCR system (384-well format) (Applied Biosystems). cDNA representing 4 ng of input total RNA was used as template in the PCR reactions. On each plate, for every sample, the GOIs and normalizer genes were tested in triplicate and a NTC was included. As expression levels of a given gene were measured across multiple 384-well plates, a plate linker sample (i.e., a sample that was run on all plates in a given study) was also included to ensure there was no plate-to-plate variability.
The normalizer genes were selected from six candidate normalizers [60S ribosomal protein L32 (rpl32), β-actin (actb), elongation factor 1-alpha 1 (ef1a1), elongation factor 1-alpha 2 (ef1a2), eukaryotic translation initiation factor 3 subunit D (eif3d) and polyadenylate-binding protein cytoplasmic 1 (pabpc1)]. Briefly, the fluorescence threshold cycle (CT) values of 48 samples (i.e., 4 individuals for each diet and treatment at each time point) were RT-qPCR-measured in duplicate for each of these transcripts using cDNA representing 4 ng of input total RNA and the 7500 Fast Real Time PCR system (Applied Biosystems), and then analyzed using geNorm. Using this software, ef1a1 (geNorm M = 0.121) and rpl32 (geNorm M = 0.154) were determined to be stably expressed in all samples tested and thus selected as normalizers.
The 384-well plate layout was designed to accommodate the simultaneous analysis of 3 genes of interest (GOIs) and the 2 normalizer genes. Each RT-qPCR experiment (i.e., each 3 GOI + 2 normalizer gene analysis) involved multiple linked plates . The completion of this research required a total of 16 independent RT-qPCR experiments. This analytical approach determined the structure of the data in the sheet Matrix non-normalized.

growth_protocol: Atlantic salmon post-smolts were fed either a high-soy oil diet (High-ω6) or a high-linseed oil diet (High-ω3) for 12 weeks. Fish holding conditions consisted of a flow-through filtered seawater system (12 L/min) and a 24-h light photoperiod; water quality was monitored daily and remained constant at 10.1 ± 0.2ºC [mean ± standard deviation (SD)] and 10.1 ± 0.5 mg O2/mL.

extract_protocol: The head kidney samples were homogenized in TRIzol using a motorized Kontes RNase-Free Pellet Pestle Grinder (Kimble Chase, Vineland, NJ, USA). Frozen homogenates were further processed by passing them through a QIAshredder (QIAGEN, Mississauga, ON, Canada) spin column following the manufacturer’s instructions. Total RNA was extracted using TRIzol (Invitrogen, Burlington, ON, Canada) following the manufacturer's instructions. Total RNA samples were treated with 6.8 Kunitz units of DNaseI (RNase-Free DNase Set, QIAGEN) with the manufacturer’s buffer (1X final concentration) at room temperature for 10 min. DNase-treated RNA samples were column-purified using the RNeasy Mini Kit (QIAGEN) following the manufacturer’s instructions. RNA integrity was verified by 1% agarose gel electrophoresis, and RNA purity was assessed by A260/280 and A260/230 NanoDrop UV spectrophotometry. Column-purified RNA samples had A260/280 ratios between 2.0 and 2.2 and A260/230 ratios between 1.8 and 2.3.

treatment_protocol:
At the end of the feeding trial, salmon were lightly anesthetized (50 mg/L MS-222) and subsequently subjected to an intraperitoneal (IP) injection –at a dose of 1 μL/g of fish (wet mass)– with either phosphate-buffered saline (PBS; Ref# 20012027, Gibco/ThermoFisher Scientific, Mississauga, ON, Canada), a solution of polyriboinosinic polyribocytidylic acid [poly(I:C); Ref# P0913, Sigma-Aldrich, Oakville, ON, Canada], or a suspension of formalin-killed A. salmonicida (Furogen Dip, Novartis, Charlottetown, PE, Canada). The poly(I:C) solution was prepared in ice-cold and 0.2 μm-filtered PBS at 2 μg/μL. The ASAL suspension was prepared in ice-cold and 0.2 μm-filtered PBS, at an optical density of 1.0 at 600 nm wavelength.
Head kidney samples were collected at 6 h or 24 h post-injection. At the sampling time points, salmon were euthanized by MS-222 overdose prior to head kidney collection. Head kidney samples were immediately flash-frozen with liquid nitrogen and stored at -80 ºC until processed for RNA extraction.

tag: antigen challenge

value: Viral mimic polyinosinic:polycytidylic acid (pIC)

tag: time point

value: 24 h post-injection

tag: diet treatment


title: HK_24h_pIC_High-w6_T6_3


title: PFC_ctrl, rep2

tag: tissue


title: One-year-old Islets - Islet 055 knockout

tag: cell type


title: One-year-old Islets - Islet 857 control

tag: genotype


title: HK_24h_pIC_High-w6_T6_6


title: One-year-old Islets - Islet 829 control


title: HK_24h_pIC_High-w6_T5_6


title: One-year-old Islets - Islet 826 control


title: One-year-old Islets - Islet 052 control


title: Embryonic Day 16.5 Pancreas - Pan 18 knockout

source_name: Pancreas

growth_protocol: For timed mating, males (Pdx1:Cre;Tff2fl/+) and females (Tff2fl/fl) were set up from 1300 to 1500 military time and copulation plugs were checked the following day from 0900 to 1100. The detection of a copulation plug was recorded as 0.5 days post-fertilization (E0.5) and the female was separated from the male. On E12.5, female mice that had gained weight compared to other littermates (presumably pregnant) were housed in a separate cage. On E16.6, pregnant dams were sacrificed, and the embryos were dissected and assessed for time stage using brightfield imaging analysis. Embryos were placed in cold 1x PBS solution, washed twice, transferred to a petri dish and serially washed in two petri dishes filled with cold 1x PBS. Internal organs of the embryos including stomach, duodenum, intestine, spleen, liver, and pancreas were dissected. Less than 12 embryos were processed within 1-hour to limit autodigestion of the pancreas. Prior to organ collection, a piece of the tail or arm was collected and washed with 1x PBS for subsequent genotyping. To avoid damage to the pancreas, it was removed in the following procedures. 1) The connective tissue linking the liver and the back of the stomach was cut first. 2) The connective tissues surrounding the intestines, starting from the duodenum, were cut. 3) The ventral pancreas was identified and the surrounding connective tissue was carefully cut, starting at the edge of the duodenum until reaching the stomach. 4) The embryonic pancreas, with ventral and the dorsal lobes together, as well as the spleen was cut and separated from the stomach and duodenum. 5) The spleen was carefully removed from the dorsal pancreas. 6) Any remaining connective tissue around the pancreas was carefully removed as much as possible. The dissected embryonic pancreas was processed immediately for subsequent RNA extraction followed by gene expression analyses.

value: Pancreas


title: HK_24h_pIC_High-w6_T5_3


title: HK_24h_pIC_High-w6_T5_1


title: One-year-old Islets - Islet 803 knockout


title: HK_24h_pIC_High-w3_T9_6

description: Challenged, 24 h post-injection, High-w3-fed fish


title: HK_24h_pIC_High-w3_T27_4


title: HK_24h_pIC_High-w3_T27_2


title: Embryonic Day 16.5 Pancreas - Pan 02 control


title: HK_24h_pIC_High-w3_T9_3


title: HK_24h_pIC_High-w3_T9_5


title: 2M-rep2, C2C12, S3

tag: cell line


title: HK_24h_pIC_High-w6_T5_4


title: HK_24h_pIC_High-w3_T9_1


title: Embryonic Day 16.5 Pancreas - Pan 19 knockout


title: HK_24h_pIC_High-w3_T27_5


title: HK_24h_pIC_High-w3_T9_2


title: ORSAM9172_WSB_9_GT20-10548_liver

city: Bar Harbor

first: Gary

last: Churchill

state: Maine

address: 600 Main St

institute: The Jackson Laboratory

data_processing:
Each tissue directory has a raw counts matrix, a vst matrix and mrna annotations table.  counts_matrix.csv: raw counts quantified by EMASE and processed by/RNAseq/Raw/GBRS/QTLViewer_setup/gathering_GBRS_total_counts.R script.  vst_matrix.csv: counts normalized by DESeq2 vst() function (quick version of variance stabilizing transformation). Matrix generated by /RNAseq/Raw/GBRS/QTLViewer_setup/QTLviewer_format.R script.  mrna_annots.csv: gene annotation file ENSEMBL version 75.
Assembly: GRCm38
Supplementary files format and content: counts_matrix.csv
Supplementary files format and content: mrna_annots.csv
Supplementary files format and content: vst_matrix.csv

source_name: liver

extract_protocol:
RNA was isolated from tissue using the MagMAX mirVana Total RNA Isolation Kit (ThermoFisher) and the KingFisher Flex purification system (ThermoFisher). Frozen tissues were pulverized using a Bessman Tissue Pulverizer (Spectrum Chemical) and homogenized in TRIzol (ThermoFisher Scientific) using a gentleMACS dissociator (Miltenyi Biotec Inc). After the addition of chloroform to the TRIzol homogenate, the RNA-containing aqueous layer was removed for RNA isolation, according to the manufacturer’s protocol, beginning with the RNA bead binding step.
2 ul of diluted (1:1000) ERCC Spike-in Control Mix 1 (Ambion by Life Technologies) was added to 100ng of each RNA sample prior to library construction. Libraries were constructed using the KAPA mRNA HyperPrep Kit (Roche Sequencing and Life Science), according to the manufacturer’s protocol. Briefly, the protocol entails isolation of polyA containing mRNA using oligo-dT magnetic beads, RNA fragmentation, first and second strand cDNA synthesis, ligation of Illumina-specific adapters containing a unique barcode sequence for each library, and PCR amplification. The quality and concentration of the libraries were assessed using the D5000 ScreenTape (Agilent Technologies) and Qubit dsDNA HS Assay (ThermoFisher), respectively, according to the manufacturers’ instructions.

treatment_protocol: Mice were sensitized on a high-fat, high-sugar (HFHS) diet

value: liver

tag: diet

value: 45percent  fat


title: ORSAM9168_B6_5_GT20-10529_liver


title: ORSAM9173_PWK_9_GT20-10562_liver


title: ORSAM9170_B6_7_GT20-10584_liver


title: HK_24h_Asal_High-w6_T6_4

value: Formalin-killed Aeromonas salmonicida (Asal)


title: ORSAM9171_B6_8_GT20-10610_liver


title: Embryonic Day 16.5 Pancreas - Pan 01 knockout


title: HK_24h_Asal_High-w6_T6_6


title: HK_24h_Asal_High-w6_T6_5


title: ORSAM9165_AJ_6_GT20-10644_liver


title: Embryonic Day 16.5 Pancreas - Pan 13 control


title: HK_24h_pIC_High-w3_T27_3


title: HK_24h_Asal_High-w6_T6_3


title: Embryonic Day 16.5 Pancreas - Pan 08 control


title: HK_24h_Asal_High-w6_T5_4


title: HK_24h_Asal_High-w6_T6_2


title: ORSAM9162_AJ_3_GT20-10558_liver


title: ORSAM9161_AJ_2_GT20-10602_liver


title: HK_24h_Asal_High-w6_T6_1


title: HK_24h_Asal_High-w6_T5_2


title: ORSAM9160_AJ_1_GT20-10641_liver


title: ORSAM9169_B6_6_GT20-10578_liver


title: ORSAM9159_NZO_4_GT20-10591_liver


title: ORSAM9158_NZO_3_GT20-10593_liver


title: ORSAM9155_129_9_GT20-10565_liver


title: ORSAM9167_AJ_8_GT20-10550_liver


title: HK_24h_Asal_High-w3_T27_4


title: HK_24h_Asal_High-w6_T5_1


title: ORSAM9157_NZO_2_GT20-10587_liver


title: HK_24h_Asal_High-w3_T9_5


title: HK_24h_Asal_High-w3_T27_2


title: HK_24h_Asal_High-w3_T27_1


title: ORSAM9163_AJ_4_GT20-10573_liver


title: ORSAM9154_129_8_GT20-10541_liver


title: HK_24h_Asal_High-w3_T9_6


title: HK_24h_Asal_High-w3_T9_3


title: ORSAM9153_PWK_7_GT20-10606_liver


title: HK_24h_Asal_High-w3_T9_4


title: HK_24h_Asal_High-w6_T5_5


title: ORSAM9151_Cast_8_GT20-10626_liver


title: ORSAM9152_PWK_6_GT20-10545_liver


title: ORSAM9149_Cast_6_GT20-10527_liver


title: ORSAM9156_NZO_1_GT20-10556_liver


title: HK_24h_Asal_High-w3_T9_2


title: HK_24h_PBS_High-w6_T6_5

description: Control, 24 h post-injection, High-w6-fed fish

value: Phosphate-buffered saline (PBS)


title: HK_24h_PBS_High-w6_T6_4


title: ORSAM9147_Cast_3_GT20-10608_liver


title: HK_24h_Asal_High-w3_T9_1


title: ORSAM9150_Cast_7_GT20-10613_liver


title: ORSAM9146_Cast_2_GT20-10634_liver


title: HK_24h_PBS_High-w6_T6_3


title: ORSAM9148_Cast_5_GT20-10627_liver


title: HK_24h_PBS_High-w6_T6_2


title: ORSAM9144_WSB_8_GT20-10617_liver


title: HK_24h_PBS_High-w6_T6_6


title: ORSAM9145_Cast_1_GT20-10537_liver


title: ORSAM9143_WSB_7_GT20-10612_liver


title: HK_24h_PBS_High-w6_T6_1


title: ORSAM9142_WSB_6_GT20-10611_liver


title: ORSAM9141_WSB_5_GT20-10643_liver


title: ORSAM9139_WSB_3_GT20-10583_liver


title: HK_24h_PBS_High-w6_T5_4


title: HK_24h_PBS_High-w6_T5_6


title: ORSAM9138_WSB_1_GT20-10589_liver


title: HK_24h_PBS_High-w6_T5_1


title: HK_24h_PBS_High-w3_T27_6

description: Control, 24 h post-injection, High-w3-fed fish


title: HK_24h_PBS_High-w6_T5_5


title: ORSAM9140_WSB_4_GT20-10625_liver


title: ORSAM9137_NOD_8_GT20-10547_liver


title: HK_24h_PBS_High-w6_T5_3


title: ORSAM9135_NOD_6_GT20-10599_liver


title: ORSAM9134_NOD_5_GT20-10569_liver


title: ORSAM9133_NOD_4_GT20-10564_liver


title: HK_24h_PBS_High-w3_T27_5


title: HK_24h_PBS_High-w3_T27_3


title: ORSAM9136_NOD_7_GT20-10581_liver


title: ORSAM9132_NOD_3_GT20-10640_liver


title: HK_24h_PBS_High-w3_T9_6


title: ORSAM9131_NOD_2_GT20-10539_liver


title: ORSAM9129_B6_4_GT20-10596_liver


title: ORSAM9130_NOD_1_GT20-10622_liver


title: HK_24h_PBS_High-w6_T5_2


title: HK_24h_PBS_High-w3_T9_4


title: ORSAM9128_B6_3_GT20-10586_liver


title: HK_24h_PBS_High-w3_T9_3


title: HK_24h_PBS_High-w3_T27_1


title: ORSAM9127_B6_2_GT20-10552_liver


title: ORSAM9126_B6_1_GT20-10536_liver


title: HK_6h_pIC_High-w6_T25_5

description: Challenged, 6 h post-injection, High-w6-fed fish

value: 6 h post-injection


title: HK_24h_PBS_High-w3_T9_2


title: ORSAM9124_129_6_GT20-10619_liver


title: HK_6h_pIC_High-w6_T25_4


title: ORSAM9125_129_7_GT20-10555_liver


title: ORSAM9123_129_4_GT20-10526_liver


title: HK_6h_pIC_High-w6_T25_1


title: HK_6h_pIC_High-w6_T25_2


title: ORSAM9122_129_3_GT20-10543_liver


title: HK_24h_PBS_High-w3_T9_5


title: HK_6h_pIC_High-w6_T23_6


title: ORSAM9120_129_1_GT20-10628_liver


title: ORSAM9121_129_2_GT20-10607_liver


title: HK_6h_pIC_High-w6_T23_4


title: ORSAM9119_PWK_5_GT20-10637_liver


title: HK_6h_pIC_High-w6_T23_2


title: HK_6h_pIC_High-w6_T25_6


title: HK_6h_pIC_High-w6_T23_3


title: ORSAM9118_PWK_4_GT20-10582_liver


title: HK_6h_pIC_High-w6_T25_3


title: ORSAM9116_PWK_2_GT20-10560_liver


title: ORSAM9117_PWK_3_GT20-10553_liver


title: HK_6h_pIC_High-w3_T22_6

description: Challenged, 6 h post-injection, High-w3-fed fish


title: HK_6h_pIC_High-w3_T22_3


title: HK_6h_pIC_High-w6_T23_1


title: ORSAM9114_NZO_8_GT20-10603_liver


title: ORSAM9112_NZO_6_GT20-10605_liver


title: HK_6h_pIC_High-w3_T4_6


title: HK_6h_pIC_High-w3_T4_5


title: HK_6h_pIC_High-w3_T22_2


title: HK_6h_pIC_High-w6_T23_5


title: ORSAM9111_NZO_5_GT20-10638_liver


title: HK_6h_pIC_High-w3_T22_4


title: PWK_9_ORSAM9110_GT20-10563_skeletal muscle

source_name: skeletal muscle

value: skeletal muscle


title: HK_6h_pIC_High-w3_T4_4


title: WSB_9_ORSAM9109_GT20-10648_skeletal muscle


title: B6_8_ORSAM9108_GT20-10585_skeletal muscle


title: HK_6h_pIC_High-w3_T4_2


title: HK_6h_pIC_High-w3_T4_3


title: B6_7_ORSAM9107_GT20-10647_skeletal muscle


title: HK_6h_pIC_High-w3_T4_1


title: ORSAM9115_PWK_1_GT20-10630_liver


title: B6_6_ORSAM9106_GT20-10623_skeletal muscle


title: AJ_8_ORSAM9104_GT20-10633_skeletal muscle


title: HK_6h_Asal_High-w6_T25_5


title: ORSAM9113_NZO_7_GT20-10577_liver


title: HK_6h_Asal_High-w6_T25_3


title: HK_6h_Asal_High-w6_T25_2


title: AJ_7_ORSAM9103_GT20-10595_skeletal muscle


title: HK_6h_Asal_High-w6_T23_6


title: HK_6h_Asal_High-w6_T23_5


title: HK_6h_Asal_High-w6_T23_4


title: HK_6h_Asal_High-w6_T23_3


title: AJ_2_ORSAM9098_GT20-10554_skeletal muscle


title: AJ_6_ORSAM9102_GT20-10535_skeletal muscle


title: AJ_3_ORSAM9099_GT20-10632_skeletal muscle


title: AJ_5_ORSAM9101_GT20-10525_skeletal muscle


title: AJ_1_ORSAM9097_GT20-10600_skeletal muscle


title: AJ_4_ORSAM9100_GT20-10621_skeletal muscle


title: NZO_4_ORSAM9096_GT20-10624_skeletal muscle


title: HK_6h_Asal_High-w6_T23_2


title: NZO_2_ORSAM9094_GT20-10594_skeletal muscle


title: NZO_3_ORSAM9095_GT20-10580_skeletal muscle


title: HK_6h_Asal_High-w3_T22_6


title: HK_6h_Asal_High-w3_T22_5


title: B6_5_ORSAM9105_GT20-10616_skeletal muscle


title: NZO_1_ORSAM9093_GT20-10631_skeletal muscle


title: HK_6h_Asal_High-w3_T22_4


title: HK_6h_Asal_High-w6_T25_1


title: 129_9_ORSAM9092_GT20-10639_skeletal muscle


title: HK_6h_Asal_High-w3_T22_2


title: 129_8_ORSAM9091_GT20-10576_skeletal muscle


title: PWK_7_ORSAM9090_GT20-10601_skeletal muscle


title: HK_6h_Asal_High-w3_T22_1


title: HK_6h_Asal_High-w3_T22_3


title: HK_6h_Asal_High-w3_T4_3


title: HK_6h_Asal_High-w3_T4_5


title: HK_6h_Asal_High-w3_T4_1


title: PWK_6_ORSAM9089_GT20-10524_skeletal muscle


title: HK_6h_PBS_High-w6_T25_6

description: Control, 6 h post-injection, High-w6-fed fish


title: Cast_8_ORSAM9088_GT20-10561_skeletal muscle


title: Cast_7_ORSAM9087_GT20-10645_skeletal muscle


title: Cast_5_ORSAM9085_GT20-10557_skeletal muscle


title: Cast_6_ORSAM9086_GT20-10551_skeletal muscle


title: HK_6h_PBS_High-w6_T25_5


title: Cast_3_ORSAM9084_GT20-10570_skeletal muscle


title: HK_6h_PBS_High-w6_T25_3


title: HK_6h_PBS_High-w6_T25_4


title: HK_6h_PBS_High-w6_T25_1


title: Cast_2_ORSAM9083_GT20-10574_skeletal muscle


title: WSB_8_ORSAM9081_GT20-10559_skeletal muscle


title: Cast_1_ORSAM9082_GT20-10615_skeletal muscle


title: WSB_7_ORSAM9080_GT20-10572_skeletal muscle


title: WSB_6_ORSAM9079_GT20-10579_skeletal muscle


title: HK_6h_PBS_High-w6_T23_4


title: HK_6h_PBS_High-w6_T23_2


title: WSB_5_ORSAM9078_GT20-10538_skeletal muscle


title: HK_6h_PBS_High-w6_T23_1


title: HK_6h_PBS_High-w3_T22_6

description: Control, 6 h post-injection, High-w3-fed fish


title: WSB_4_ORSAM9077_GT20-10549_skeletal muscle


title: WSB_1_ORSAM9075_GT20-10636_skeletal muscle


title: NOD_7_ORSAM9073_GT20-10534_skeletal muscle


title: HK_6h_PBS_High-w3_T22_4


title: HK_6h_PBS_High-w3_T22_3


title: HK_6h_PBS_High-w3_T4_6


title: WSB_3_ORSAM9076_GT20-10571_skeletal muscle


title: HK_6h_PBS_High-w3_T22_1


title: HK_6h_PBS_High-w6_T23_6


title: NOD_5_ORSAM9071_GT20-10629_skeletal muscle


title: NOD_3_ORSAM9069_GT20-10532_skeletal muscle


title: HK_6h_PBS_High-w3_T4_5


title: HK_6h_PBS_High-w6_T23_5


title: NOD_4_ORSAM9070_GT20-10575_skeletal muscle


title: HK_6h_PBS_High-w3_T4_3


title: Day 7-T-F3

city: Jeongeup

first: Yoon

last: Cho

state: Jeollabuk-do

address: 30, Baekhak 1-gil

country: South Korea

institute: Korea Institute of Toxiology

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and filtered for low-complexity or low-quality sequence(using fastp), then mapped to dm6 (NCBI) whole genome using STAR.
Read count extraction using HTSeq-count.
Assembly: dm6
Supplementary files format and content: tab-delimited text files include read count values for each Sample

source_name: Whole

extract_protocol:
RNA was harvested using Trizol reagent (Invitrogen). 500 ng of total RNA was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using LEXOGEN Quant-Seq Library Prep Kit (Cat#015) standard protocols.

treatment_protocol: The flies of both sexes were exposed to CMIT/MIT aerosol for 1 hour at 330 mg/m3.

value: Whole

value: WT

tag: treatment

value: Inhalation, CMIT/MIT, 330 mg/m3


title: HK_6h_PBS_High-w3_T22_5


title: HK_6h_PBS_High-w3_T4_4


title: NOD_2_ORSAM9068_GT20-10598_skeletal muscle


title: NOD_1_ORSAM9067_GT20-10544_skeletal muscle


title: Day 7-T-F2


title: HK_6h_PBS_High-w3_T4_1


title: Day 7-T-F1


title: B6_4_ORSAM9066_GT20-10620_skeletal muscle


title: B6_3_ORSAM9065_GT20-10588_skeletal muscle


title: B6_2_ORSAM9064_GT20-10523_skeletal muscle


title: Day 7-T-M1


title: Day 7-T-M2


title: B6_1_ORSAM9063_GT20-10568_skeletal muscle


title: 129_7_ORSAM9062_GT20-10597_skeletal muscle


title: Day 7-VC-F3

value: Inhalation, Filtered air


title: Day 7-VC-F1


title: 129_6_ORSAM9061_GT20-10566_skeletal muscle


title: Day 7-VC-M3


title: Day 7-VC-F2


title: 129_3_ORSAM9059_GT20-10635_skeletal muscle


title: 129_4_ORSAM9060_GT20-10618_skeletal muscle


title: 129_2_ORSAM9058_GT20-10542_skeletal muscle


title: 129_1_ORSAM9057_GT20-10642_skeletal muscle


title: Day 2-T-F3


title: PWK_4_ORSAM9055_GT20-10609_skeletal muscle


title: Day 2-T-F2


title: Day 7-VC-M2


title: Day 2-T-F1


title: Day 7-T-M3


title: PWK_2_ORSAM9053_GT20-10592_skeletal muscle


title: Day 2-T-M3


title: Day 2-T-M2


title: PWK_1_ORSAM9052_GT20-10533_skeletal muscle


title: NZO_8_ORSAM9051_GT20-10531_skeletal muscle


title: Day 2-T-M1


title: NZO_7_ORSAM9050_GT20-10590_skeletal muscle


title: Day 2-VC-F3


title: NZO_6_ORSAM9049_GT20-10528_skeletal muscle


title: Day 2-VC-F2


title: 100_GES15_02185_Islet

source_name: Islet

extract_protocol:
RNA was isolated from pancreatic islets using the RNeasy Micro kit (Qiagen), according to manufacturers’ protocols, including the optional DNase digest step. RNA concentration and quality were assessed using the Nanodrop 8000 spectrophotometer (Thermo Scientific) and the RNA 6000 Pico or RNA ScreenTape assay (Agilent Technologies)
2 ul of diluted (1:1000) ERCC Spike-in Control Mix 1 (Ambion by Life Technologies) was added to 100ng of each RNA sample prior to library construction. Libraries were constructed using the KAPA mRNA HyperPrep Kit (Roche Sequencing and Life Science), according to the manufacturer’s protocol. Briefly, the protocol entails isolation of polyA containing mRNA using oligo-dT magnetic beads, RNA fragmentation, first and second strand cDNA synthesis, ligation of Illumina-specific adapters containing a unique barcode sequence for each library, and PCR amplification. The quality and concentration of the libraries were assessed using the D5000 ScreenTape (Agilent Technologies) and Qubit dsDNA HS Assay (ThermoFisher), respectively, according to the manufacturers’ instructions.

value: Islet


title: Day 2-VC-F1


title: Day 2-VC-M3


title: Day 7-VC-M1


title: 99_GES15_02184_Islet


title: 98_GES15_02183_Islet


title: 97_GES15_02182_Islet


title: Day 2-VC-M1


title: 96_GES15_02181_Islet


title: PWK_3_ORSAM9054_GT20-10604_skeletal muscle


title: Day 1-T-F3


title: Day 1-T-F2


title: cell culture with 1000uM IAA sample 2

city: Little Rock

first: Zulema

last: Udaondo

state: AR

address: 501 Jack Stephens Drive

department: Biomedical Informatics

institute: UAMS

data_processing:
Libraries were sequenced on the Illumina NextSeq 500 sequencer. Initial quality control checks were carried out using FastQC software on the raw sequences. Alignment of reads to reference genome was done using STAR software and assembled using StringTie2. Raw counts of aligned transcripts were processed using DESeq2 software using R.
Assembly: GCA_001606165.1
Supplementary files format and content: Comma-delimited text file with raw counts for each sample

organism: Serratia plymuthica

source_name: rhizosphere of wheat

growth_protocol: S. plymuthica A153 was grown in minimal medium with glucose as sole carbon source in the absence and presence of 0.25 and 1 mM IAA. RNA-seq samples were collected at the beginning of the stationary phase of growth, stage at which the expression of the andrimid gene cluster was maximal.

extract_protocol:
RNA sequencing was done at the GENYO Research Center (Granada, Spain). Prior to preparation of the RNA library, ribosomal RNAs were removed from the samples using the RiboZeroTM kit (Epicentre, Gram-Negative-Bacteria#MRZGN126) following the manufacturers’ instructions.
Samples were processed with the TruSeq Stranded Total RNA Library Prep Kit (Illumina) following the provided sample preparation guide. The final library (adapter and index included) was validated using the DNA-specific chip Agilent DNA 1000.

value: rhizosphere of wheat

value: pure culture

value: 1000uM IAA


title: 95_GES15_02180_Islet


title: Day 1-T-F1


title: cell culture with 1000uM IAA sample 3


title: cell culture with 1000uM IAA sample 1


title: cell culture with 250uM IAA sample 3

value: 250uM IAA


title: Day 1-T-M3


title: cell culture with 250uM IAA sample 2


title: 93_GES15_02178_Islet


title: cell culture with 250uM IAA sample 1


title: NZO_5_ORSAM9048_GT20-10567_skeletal muscle


title: Day 1-T-M2


title: cell culture control 3

value: Control


title: Day 2-VC-M2


title: Day 1-T-M1


title: Day 1-VC-F3


title: cell culture control 1


title: Δfim LB3

first: Xiamei

last: Kang

address: No. 388, Yuhangtang Road

institute: Zhejiang University

description: Δfim LB3

data_processing:
Fastp v.0.19.7 was used to process the raw data in fastq format to get clean data (parameter qualified_quality_phred 15；cut_mean_quality 20；length_required 75).
Clean reads were mapped to the NCBI Rfam database, and rRNA sequences were removed by Bowtie2(v2.33)（parameter default）.
Sequence reads after ribosome removal were aligned to the Homo sapiens reference genome GRCh38 from ensembl database by Hisat2 (v2.1.0)（parameter default）.
Salmon software v1.2.1 was used to obtain the expression for each gene（parameter default）.
EdgeR software v3.20.2 was used to obtain the differentially expressed genes（parameter fold change>2;FDR<0.05）.
Assembly: GenBank accession number CP068386.1 (chromosome) and two plasmids (CP068387.1 and CP068388.1)
Supplementary files format and content: Tab_delimited txt file include raw counts for each sample

organism: Salmonella enterica subsp. enterica serovar Pullorum

extract_protocol:
The total RNA of all strains was extracted with Trizol reagent, assessed using Thermo NanoDrop One and Agilent 4200 Tape Station instruments.
The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina was used for cDNA library construction

value: fim knockout

value: egg white


title: 90_GES15_02175_Islet

value: 45percent  fat


title: Δfim LB1

description: Δfim LB1


title: 89_GES15_02174_Islet


title: Day 1-VC-F2


title: Δfim LB2

description: Δfim LB2


title: Δfim egg white2

description: Δfim egg white2

value: LB


title: 94_GES15_02179_Islet


title: cell culture control 2


title: 92_GES15_02177_Islet


title: Δfim egg white3

description: Δfim egg white3


title: Day 1-VC-F1


title: Day 1-VC-M3


title: Δfim egg white1

description: Δfim egg white1


title: WT egg white3

description: WT egg white3

value: wild type


title: WT egg white2

description: WT egg white2


title: 86_GES15_02171_Islet


title: WT egg white1

description: WT egg white1


title: 85_GES15_02170_Islet


title: WT LB3

description: WT LB3


title: Day 1-VC-M2


title: WT LB2

description: WT LB2


title: Day 1-VC-M1


title: 91_GES15_02176_Islet


title: WT LB1

description: WT LB1


title: 84_GES15_02169_Islet


title: C. auris_60min_SPB00525 _R2

organism: Candidozyma auris

source_name: WT control C. auris cells in SC medium treated with SPB00525 for 60 min_rep2

value: Yeast cells

value: 60 min in SC medium + 6 ug/ml SPB00525


title: Naive control rep 4

city: Houston

first: Navin

last: Varadarajan

state: TX

address: 4722 Calhoun Rd

department: Chemical and Biomolecular Engineering

institute: University of Houston

data_processing:
Sequence reads were trimmed for adaptor sequence and paired using Trimmomatic v0.39
Aligning paired reads to the Syrian golden hamster genome (MesAur 1.0, GCF_000349665.1) and extraction of read counts were performed using STAR v2.7.9a
Assembly: MesAur 1.0
Supplementary files format and content: tab-delimited text files include raw counts for all samples

organism: Mesocricetus auratus

source_name: Lung

growth_protocol: Syrian golden hamster lungs cells were rested for 5-6 hours in RPMI supplemented with 10% fetal bovine serum (FBS)  with 5% CO2 at 37C before proceeding for RNA isolation

extract_protocol:
Total RNA was extracted from whole lung cells using RNeasy kit (Qiagen, #74104), following the manufacturer's instructions. Extracted RNA was DNase treated using a DNA-free DNA removal kit (Invitrogen, #AM1906).
mRNA library preparation (poly A enrichment)

treatment_protocol: 4 Syrian golden hamsters were intransally treated with 60µg of NanoSTING for four consecutive days. 4 naive hamsters were used as controls

value: Lung

value: No treatment


title: C. auris_60min_Control_R2

source_name: WT control C. auris cells in SC medium for 60min_rep2

value: WT

value: 60 min in SC medium


title: 88_GES15_02173_Islet

value: Islet


title: C. auris_60min_SPB00525 _R1

source_name: WT control C. auris cells in SC medium treated with SPB00525 for 60 min_rep1


title: C. auris_15min_SPB00525 _R2

source_name: WT control C. auris cells in SC medium treated with SPB00525 for 15 min_rep2

value: 15 min in SC medium + 6 ug/ml SPB00525


title: 82_GES15_02167_Islet


title: 83_GES15_02168_Islet


title: C. auris_15min_SPB00525 _R1

source_name: WT control C. auris cells in SC medium treated with SPB00525 for 15 min_rep1


title: C. auris_60min_Control_R1

source_name: WT control C. auris cells in SC medium for 60min_rep1


title: C. auris_15min_Control_R2

source_name: WT control C. auris cells in SC medium for 15min_rep2

value: 15 min in SC medium


title: 81_GES15_02166_Islet


title: C. auris_15min_Control_R1

source_name: WT control C. auris cells in SC medium for 15min_rep1


title: Naive control rep 2


title: MCF7_TAM10_Tamoxifen_Rep2

first: Luciana

middle: Ines

last: Gomez Acuna

institute: MRC-University of Edinburgh

data_processing:
Capture-C data were aligned to the hg19 genome and processed using distiller-nf 0.3.3 (https://doi.org/10.5281/zenodo.3350937) with default settings.
Coolers filtered for q>=30 were used and ICE balanced (Imakaev et al., 2012) using the cooler (Abdennur et al., 2020) command 'cooler balance --cis_only --blacklist blacklist.bed' where blacklist.bed contains all regions outside of the capture probe area.
Assembly: hg 19
Supplementary files format and content: Cooler files
Library strategy: Capture-C-seq

growth_protocol: MCF7 cells were maintained in DMEM supplemented with 10% fetal calf serum (FCS) and antibiotics in humidified atmosphere with 5% CO2 at 37C. Before treatment, cells were grown in phenol red-free DMEM supplemented with 10% charcoal stripped FCS for 4 days.

extract_protocol:
Cells were fixed with 1 .5 % formaldehide for 10 minutes. After lisis, nuclei were subjeted to in situ DpnII restriction and ligation. DNA was decrosslinked and extracted with phenol:chloroform. Regions of interest were captured from 3C libraries using fragmented and biotinilated tiled BACs. Captured regions' coordenates (hg19): chr2:11450601-11945472; chr21:16143548-17096678
Before Capture, 3C libraries were prepaired with NEBNext® Ultra™ II DNA Library Prep for Illumina

treatment_protocol:
Cells were treated with either vehicle (ethanol) or E2 10nM for 5, 30 or 60 minutes.
WT, GREB1 enhacner deletion or NRIP1 enhancer deletion were treated with either vehicle (ethanol) or E2 10nM for 30 minutes.
Cells were treated with either vehicle (ethanol), Tamoxifen 10 uM or  E2 10nM 30 minutes.
Cells were treated with either vehicle (DMSO), Flavopiridol 10 μM or Triptolide 10 μ for 5 min and then, with either vehicle (ethanol) or E2 10nM for additional 30 minutes.

value: Mammary gland, epithelial

value: Tamoxifen 10 uM 30 min


title: 79_GES15_02164_Islet


title: NanoSTING rep 4

value: NanoSTING


title: NanoSTING rep 3


title: MCF7_TAM10_Vehicle_Rep2

value: Vehicle


title: MCF7_TAM10_Tamoxifen_Rep1


title: MCF7_TAM10_E2_Rep1

value: E2 10 nM 30 min


title: NanoSTING rep 2


title: MCF7_TAM10_Vehicle_Rep1


title: Naive control rep 3


title: Naive control rep 1


title: MCF7_TAM10_E2_Rep2


title: 76_GES15_02161_Islet

value: 45percent  fat


title: 80_GES15_02165_Islet


title: 78_GES15_02163_Islet


title: 73_GES15_02158_Islet


title: WT_siC_unt_1

city: Cambridge

first: Ana

last: Tufegdzic Vidakovic

state: Cambridge

address: Cambridge Biomedical Campus, Francis Crick Ave

institute: MRC-LMB

data_processing:
*library strategy: transient transcriptome sequencing (TT-seq)
TTchem-seq reads were trimmed and quality-filtered with Trim Galore, using a quality threshold of 30. Trimmed reads were aligned to the hg38 and Saccharomyces cerevisiae (sacCer3) genomes using STAR aligner90 with basic two-pass mapping.
PCR duplicates were marked and removed using Picard, and the correlation between replicates was checked using deepTools multiBamSummary. Replicates were merged, and resulting BAM files were split by strand. Bigwig files were created using deepTools bamCoverage, normalising to the number of spike-in reads.
Assembly: GRCh38
Supplementary files format and content: bigWig

source_name: Flp-In T-REx HEK293

growth_protocol: DMEM (31966047), 10% fetal bovine serum, 1% penicillin/streptomycin. Cells were cultured at 37°C with 5% CO2.

extract_protocol:
RNA was extracted by TRIzol (Thermo Fisher Scientific, 15596026) accordingly to the manufacturers instructions. As a control for equal sample preparation, we spiked-in S. cerevisiae (strain BY4741, MATa, his3D1, leu2D0, met15D0, ura3D0) 4-thiouracil (4TU)-labelled RNA. For purification of 4sU labelled RNA, 100 ug mammalian 4SU labelled RNA was spiked-in with 1ug of 4tU-labelled S. cerevisiae RNA. The 101 ug RNA was fragmented by addition of 20 uL of 1 M NaOH to fragment RNA and left on ice for 20 min. Fragmentation was stopped by addition of 80 uL 1 M Tris pH 6.8 and cleaned up twice with Micro Bio-Spin‚P-30 Gel Columns (BioRad, 7326223). Biotinylation of 4sU-residues were carried out in a total volume of 250 ul, containing 10 mM Tris-HCl [pH 7.4], 1 mM EDTA, 50uL DMF and 5mg MTSEA biotin-XX linker (Biotium, BT90066) for 30 min at room temperature in the dark. RNA was then purified by phenol:chloroform extraction, denatured by 10 min incubation at 65C and added to 200 uL uMACS Streptavidin MicroBeads (Miltenyi, 130-074-101). RNA was incubated with beads for 15 min at room temperature and beads applied to a uColumn in the magnetic field of a uMACS magnetic separator. Beads were washed twice with pull-out wash buffer (100 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1 M NaCl and 0.1% Tween20). 4sU-RNA was eluted twice by addition of 100 mM DTT and RNA cleaned up using the RNeasy MinElute kit (Qiagen, 74204).
Libraries for RNA sequencing were prepared using the KAPA RNA HyperPrep Kit (KR1350) with modifications. 75 ng of RNA per sample were mixed with FPE Buffer, but fragmentation procedure was omitted and RNA was instead denatured at 65C for 5 min. The rest of the procedure was performed as recommended by the manufacturer, with the exception of SPRI bead purifications: after adaptor ligation, 0.95x and 1x SPRI bead-to-sample volume ratios were used (instead of two rounds of SPRI purification with 0.63x volume ratios). This was done to retain smaller (150-300 bp) cDNA fragments in the library which would otherwise be lost in size selection. The libraries were quality controlled by electrophoresis on a Tapestation system (Agilent), quantified by Qubit (Thermofisher).

treatment_protocol: For each condition, 2 wells of a 6-well plate were seeded, each containing 600,000 cells, and transfected with siRNAs (Dharmacon, L-020270-02 and D-001810-10) the next day, using Lipofectamine RNAiMax (Invitrogen, 13778100), according to manufacturers instructions. 40 nM final siRNA concentration was used. 24 hours after transfection, cells of the same condition were re-seeded and combined into a single 10cm dish. The following day, cells were treated with 300nM triptolide for2 hours or vehicle, and nascent RNA was in vivo labelled with a 1 mM 4sU (Glentham Life Sciences, GN6085) pulse for exactly 15 min (e.g. for 2-hour triptolide time point samples, 4sU was added 1 h and 45 min after treatment).

value: Flp-In T-REx HEK293

value: Parental WT

value: siControl


title: WT_siInts8_unt_1


title: WT_siC_TPL_3

value: siControl, triptolide


title: WT_siInts8_unt_2


title: WT_siC_unt_3


title: 74_GES15_02159_Islet


title: fyn_binding_output1

last: Escobedo

state: Catalonia

address: Carrer Dr Aiguader 88

department: Systems and synthetic biology

institute: CRG

description: FYN-SH3_core_DTS_binding_scores_PRD1super.csv

data_processing:
DiMSum (Demultiplex with Cutadapt)
DiMSum (QC raw reads with FastQC)
DiMSum (Trim reads with Cutadapt)
DiMSum (Process and analyse variants with DiMSum)
Assembly: NA
Supplementary files format and content: All in tabular format. sparse_DTS_core_mutagenesis_scores.csv contains a list of variants, each linked to the protein they are related to (Protein), its Uniprot identifier (UniprotCode), their complete amino acid sequence (aa_seq), the sequence in core positions (core), the hamming distance to the WT protein they are related to (Nham_aa), whether they were used as the reference in DiMSUM processing (WT), and their fitness and error values as obtained directly from DiMSum (fitness_raw and sigma_raw) and after scaling in the 0-1 range (fitness_scaled and sigma_scaled). The FYN-SH3_core_DTS_binding_scores_PDR1super.csv file contains equivalent aa_seq, core, Nham_aa, WT, fitness_scaled, sigma_scaled, fitness_raw and sigma_raw fields for variants all related to FYN SH3 binding to PDR1super. The FYN-SH3_core_permissivity_scores contains equivalent aa_seq, WT, fitness_scaled, sigma_scaled, fitness_raw and sigma_raw fields for variants in the permissivity library.
Library strategy: PCR amplicon

organism: Saccharomyces cerevisiae

source_name: yeast cells

growth_protocol: The bottlenecked library pool containing randomly subsampled fractions of the FYN SH3, CI-2A and CspA DTS core libraries in pGJJ162, the full complexity FYN SH3 DTS core library in pGJJ159-PRD1super and the FYN SH3 permissivity library in pGJJ162 were transformed in BY4741 yeast cells by heat shock. The number of transformation reactions was scaled depending on the final selection volume, which was in turn scaled to achieve a 100X coverage at the most stringent selection step in terms of cell count bottlenecking (inoculation in selection medium, see below). An individual transformation was performed per 200 mL of selection culture. For each individual transformation, 25 mL YPDA were inoculated with BY4741 colonies from a freshly streaked YPDA plate and grown overnight at 30ºC. The process was performed in parallel for each independent biological replicate, starting from a different BY4741 colony (2 replicates for the subsample DTS core library pool, 3 replicates for the other experiments). The precultures were used to inoculate 180 mL of YPDA to an OD600 of 0.3, which was grown for 4 h at 30ºC. Cells were harvested by 5 min centrifugation at 3,000 g, washed in water and treated with 8.6 mL SORB for 30 min. 3.5 μg of plasmid DNA were then added along with 175 μL of 10 mg/mL sonicated salmon sperm DNA (Santa Clara, CA, US), and the mixture was agitated at room temperature for 10 minutes followed by addition of 35 mL of plate mixture plus another 30 min incubation. 3.5 mL of DMSO were then added and the mixture was heat-shocked at 42ºC for 20 min. After the heat-shock, cells were harvested by 5 min centrifugation at 3,000 g, resuspended in recovery media and incubated at 30ºC for 1 h. Cells were then harvested again and resuspended in 200 mL of SC -URA selective medium. A 10 μL aliquot was serially diluted and plated in SC -URA to assess transformation efficiency.  All biological replicate transformation cultures were grown in parallel at 30ºC for ~24 h. Then, the selection input cultures were inoculated by resuspending water washed harvested cell pellets in SC -URA -ADE to an OD600 of 0.1 (800 mL in 5 L flasks in the cases of the subsampled DTS core library pool and FYN SH3 binding library, 400 mL in 2L flasks in the case of the FYN SH3 permissivity library). The input cultures were grown at 30ºC and harvested upon reaching an OD600 of 1.6 (5 generations), which typically occurred from 12 to 15 h after inoculation. Cell pellets were stored at -20ºC. Aliquots of the input culture were used to inoculate the selection (output) cultures (SC -URA -ADE, 200 μg/mL methotrexate) at an OD600 of 0.05. The output cultures were grown at 30ºC for ~30 h (subsampled DTS core library pool and FYN SH3 binding library) or until they reached an OD600 of 1.6. At that time, cells were harvested by 5 min centrifugation at 3,000 g, thoroughly washed in water and stored at -20ºC.

extract_protocol:
Input and output culture cell pellets were resuspended in DNA extraction buffer (10 mM Tris-HCl, 100 mM NaCl, 2% Triton-X, 1% SDS, pH 8). Volumes were scaled depending on the volume of cell pellets to process. For pellets from 100 mL cultures harvested at OD600 1.6, 1 mL of DNA extraction buffer was used. Cells were lysed by cyclically freezing in liquid nitrogen followed by incubation in a 62ºC water bath twice. Total DNA was extracted by adding 1 mL of a 25:24:1 phenol:chloroform:isoamyl alcohol mixture (Merck, Darmstadt, Germany) along with 1 g of acid washed glass beads (Sigma-Aldrich, Saint Louis, MO, US) and vortexing for 10 min. The mixture was centrifuged at room temperature for 30 min at 3,300 g and the aqueous phase (upper layer) was recovered in a separate tube. 1 mL of the phenol:chloroform:isoamyl alcohol was added to it and the mixture was vortexed for 2 min followed by 45 min centrifugation at 3,300 g. The aqueous phase was again transferred to a new tube and mixed with 0.1 volumes of 3 M sodium acetate and 2.2 volumes of pure ethanol previously cooled down at -80ºC. The mixture was kept at -20 ºC for 30 min and spun down at 3,300 g for 30 min at 4ºC. The DNA pellets were dried overnight in a fume extraction hood.  Plasmid DNA from total DNA extracts was quantified by qPCR using the oGJJ152-oGJJ153 oligo pair that anneals at the origin of replication of all pGJJ vector series. The qPCR reaction was performed in a LightCycler 400 instrument (Roche, Basel, Switzerland) using the SYBR green qPCR 2X master mix from Thermo Fisher (Waltham, MA, US). Quantification was achieved by measuring a standard curve on a serial dilution of a control sample of known concentration in the same qPCR run as the query samples, and subtracting blank measurements.  Library preparation for sequencing in Illumina instruments followed a two-step PCR protocol. Frameshifts and a segment of the Illumina adapter overhangs were introduced in PCR1. For each sample, 8x50 μL PCR1 reactions were performed using 31.25 million plasmid molecules as a template as quantified by qPCR. PCR1 used the oGJJ595 and the oGJJ748 frameshifting oligo pools and consisted of 11 cycles. Next, all reactions corresponding to the same sample were pooled and treated with 16 μL ExoSAP for 30 min at 37ºC, then for 20 min at 80ºC. Samples were cleaned-up using MinElute columns, eluted in 30 μL of pre-warmed water (55ºC) and 1.5 μL were used as template for each PCR2 reaction.16x50 μL PCR2 reactions were performed per sample to introduce the rest of the Illumina adapters and the dual index barcodes. PCR2 reactions corresponding to the same sample were pooled, concentrated using a MinElute column, and run in a 2% agarose gel. Bands of the correct sizes were excised from the gel and sequencing-ready DNA was recovered by using a centrifugal filter unit followed by MinElute clean-up. Samples were quality controlled by the CRG Genomics core facility using a TapeStation instrument (Agilent, Santa Clara, CA, US), quantified by qPCR and sequenced in either a NextSeq500 (FYN SH3 permissivity library) or NextSeq2000 (subsampled core DTS library pool and FYN SH3 binding library) instrument (Illumina, San Diego, CA, US).
Combinatorial core libraries using a reduced alphabet of hydrophobic amino acids (F, L, I, M, V) were built by multiple segment Gibson assembly of oligonucleotides bearing the DTS degenerate codon at core residue-encoding positions (oligos used in this study were all obtained from IDT, Coralville, IA, US). Each of the genes encoding the FYN SH3 domain, CI-2A and CspA were codon optimized for expression in Saccharomyces cerevisiae and divided in two segments smaller than 200 bp each, which was the size limit for commercial synthetic degenerate oligo production at the time of performing this study. Both the 5’ and the 3’ gene segments contained a homology region of at least 25 bp to the linearized pGJJ191 mutagenesis vector, plus an internal homology region of at least 25 bp. Double stranded Gibson assembly inserts were produced from single stranded commercial oligonucleotides via a single cycle PCR reaction using a 3’ annealing reverse primer specific to each segment (see Table ). The pGJJ191 vector was linearized via a 25-cycle PCR reaction using primers oGJJ311 and oGJJ406, followed by 1h DpnI (NEB) treatment at 37ºC plus inactivation at 80ºC for 20 minutes (1 μL enzyme per 50 μL PCR reaction). Both inserts and vector PCR reactions were cleaned-up in MinElute columns (Qiagen, Hilden, Germany). Gibson assembly mixtures contained 300 ng of linearized pGJJ191 plus the two insert segments at a 5:1 molar ratio each, along with a Gibson enzyme mixture produced in house by the Protein Technologies core facility at the CRG.  Conversely, the FYN SH3 permissivity library was purchased as an oligo pool from Twist Bioscience (San Francisco, CA, US). The oligo pool was amplified in a 13 cycle PCR reaction with primers oAE018 and oAE227, which was cleaned up using a MinElute column. The insert amplified oligo pool contained 25 bp homology regions at both ends and was mixed at a 5 to 1 molar ratio with 300 ng of linearized pGJJ191 and subsequently with the Gibson enzyme mixture.  All Gibson assembly reactions were incubated at 50ºC for 8h, dialyzed against water using 0.025 μm filters (Millipore, Burlington, MA, US) for 1.5h, concentrated using a SpeedVac and transformed into E. coli C3020 cells (NEB, Ipswich, MA, US). The number of transformants was assessed by colony count in serial dilutions of transformation outgrowth aliquots in LB plates supplemented with 50 μg/mL spectinomycin, and 1 to 5 equivalent transformation reactions were pooled to reach at least 20X variant coverage per library as required. Plasmid DNA was extracted using the Qiagen Plasmid Plus Midi kit and the library inserts were obtained through double endonuclease digestion (HindIII-HF and NheI-HF, NEB) followed by agarose gel purification achieved by a combined use of centrifugal filter units (Millipore) and MinElute clean-up. Inserts were introduced in double-restricted, Quick CIP-trated (NEB) pGJJ162 (abundancePCA) or pGJJ159-PRD1super (bindingPCA) vectors by overnight thermal-cyclic ligation using T4 DNA ligase. The pGJJ159-PRD1super vector was previously obtained by introducing the S. cerevisiae codon-optimized PRD1super encoding gene from commercial oligo synthesis (IDT) into BamHI-SpeI double restriction-linearized pGJJ159 via Gibson assembly. Ligation mixtures were dialyzed against water, SpeedVac-concentrated and transformed into E. coli C3020 electrocompetent cells. Equivalent transformations were combined to reach at least 20X variant coverage, and selection-ready libraries in PCA vectors were Midi-prep extracted.  For sparse sampling of combinatorial core libraries (FYN SH3, CI-2A and CspA in pGJJ162), the full-complexity libraries were transformed into E. coli C3020 electrocompetent cells and bottlenecked by outgrowth medium serial dilution into overnight selective medium (LB supplemented with 50 μg/mL ampicillin) assessed in parallel through colony count of plated aliquots, aiming at ~10,000 transformants per library. Bottlenecked libraries were extracted by mini-prep, qPCR quantified and mixed to equivalent molar ratios.

treatment_protocol: abundancePCA and bindingPCA (High-resolution mapping of protein concentration reveals principles of proteome architecture and adaptation, Levy and Michnick, 2014; Mapping the energetic and allosteric landscapes of protein binding domains, Faure et. Al, Nature 2022)

tag: lab strain

value: yeast cells


title: fyn_binding_output2


title: fyn_binding_output3


title: WT_siC_unt_2


title: WT_siC_TPL_1


title: KO_siInts8_unt_3

value: ARMC5 KO


title: WT_siInts8_unt_3


title: fyn_binding_input3


title: KO_siInts8_unt_2


title: fyn_binding_input2


title: 70_GES15_02155_Islet


title: 77_GES15_02162_Islet


title: KO_siInts8_unt_1


title: 75_GES15_02160_Islet


title: NanoSTING rep 1

value: Lung


title: WT_siC_TPL_2


title: 69_GES15_02154_Islet


title: Pol II NET-seq, MnCl2-treated, replicate 3

first: Abigail

last: Huffines

address: 720 20th St S

description: MnClC

data_processing:
*library strategy: NET-seq
Base-calling was conducted with bcl convert software.
Deduplication based on the UMI sequence was performed via fqtrim (version 0.9.7) with the "-C" option.
First trimming step was achieved via cutadapt (version 3.4) and the "-g -no-indels" parameters. Reads containing the 5' sequence 5'-AGNNNNNNNNTG-3' were isolated, and this sequence was removed.
Second trimming step was performed with cutadapt (version 3.4) and the "-a -no-indels" parameters. The 3' sequence 5'-CTGTAGGCACCAT-3' was removed from reads.
Reads were aligned to the Saccharomyces cerevisiae genome via STAR (version 2.7.1a). The STAR alignIntronMax parameter was set to 1.
Output BAM files from alignment were indexed using SAMtools (version 1.6).
The bamCoverage tool from the deepTools package was used to generate bigWig files, with parameters set to "--Offset 1 --normalizeUsing CPM".
The computeMatrix tool (scale-regions subtool) from the deepTools package with the parameters set to "-b 3000 -a 3000 --regionBodyLength 5000 --skipZeros" was used.
The plotProfile tool from the deepTools package was used to visualize data.
Assembly: S. cerevisiae R64-1-1
Supplementary files format and content: Processed data for each sample are included in an individual bigWig (.bw) file.

growth_protocol: Yeast were grown at 30ºC with nutation.

extract_protocol:
Cell harvest, lysis, and RNA extraction were performed exactly the same as described in previous publications from our lab. To summarize, yeast cells were rapidly harvested via filtration and lysed under cryogenic conditions. Pol II was isolated via immunoprecipitation and an acidic phenol/chloroform extraction was used to extract total RNA.
NET-seq library construction was performed exactly as described in previous publications from our lab. To summarize, a UMI-containing DNA linker was ligated onto the 3' end of RNAs after the total RNA extraction. Next, reverse transcription was performed and resulting DNAs were circularized. Finally, circular DNAs were amplified, libraries were purified via PCRClean DX beads, and reads were sequenced with the Illumina NovaSeq 6000 machine.

treatment_protocol: Immediately prior to the immunoprecipitation, frozen cell lysates were resuspended in lysis buffer (5 mL * grindate weight; previously described in Clarke et al, PNAS (2018)). For CaCl2 treated samples, a final concentration of 5 mM of CaCl2 was added, then samples were mixed thoroughly and incubated at 37ºC for 2 minutes. To quench the reaction, a final concentration of 5 mM of EDTA (pH 8.5) was added. For the MnCl2 treated samples, a final concentration of 10 mM MnCl2 was added and samples were mixed well. Samples were incubated on ice for 20 minutes, then the reaction was quenched with the addition of 5 mM EDTA (pH 8.5). Finally, samples were incubated on ice for 30 minutes.


title: fyn_binding_input1


title: 72_GES15_02157_Islet


title: KO_siC_unt_3


title: Pol II NET-seq, MnCl2-treated, replicate 2

description: MnClB


title: sparse_DTS_cores_output_1


title: Pol II NET-seq, CaCl2-treated, replicate 3

description: CaClC


title: KO_siC_unt_2


title: sparse_DTS_cores_output_2


title: 66_GES15_02151_Islet


title: 67_GES15_02152_Islet


title: 68_GES15_02153_Islet


title: KO_siC_unt_1


title: Pol II NET-seq, MnCl2-treated, replicate 1

description: MnClA


title: sparse_DTS_cores_input_2


title: Pol II NET-seq, CaCl2-treated, replicate 2

description: CaClB


title: KO_siC_TPL_3


title: KO_siC_TPL_2


title: sparse_DTS_cores_input_1


title: Pol II NET-seq, CaCl2-treated, replicate 1

description: CaClA


title: 65_GES15_02150_Islet


title: NOD_8_ORSAM9074_GT20-10646_skeletal muscle

value: skeletal muscle